Hydrophobic Molecule-Induced Branched Polymer Aggregates and their Use

ABSTRACT

Symmetrically and asymmetrically branched homopolymers are modified at the surface level with functional groups that enable forming aggregates with water insoluble or poorly water soluble pharmaceutically active agents (PAA). The aggregates formed are specifically induced by interaction of PAA and homopolymer and are different from aggregates that are formed by the polymer alone in the absence of the PAA or by the PAA alone in the absence of the polymer. Such aggregates can be used to improve drug solubility, stability, delivery and efficacy.

The instant application claims benefit to U.S. Ser. No. 61/431,042 filed9 Jan. 2011, U.S. Ser. No. 61/500,633 filed 24 Jun. 2011, and U.S. Ser.No. 61/502,793 filed 29 Jun. 2011, the content of each of which isincorporated herein by reference in entirety.

FIELD

The present disclosure relates to a surface modified branched polymer(MBP), which can either be a surface modified symmetrically branchedpolymer (SBP) or a surface modified asymmetrically branched polymer(ABP), which on exposure to a water insoluble or poorly water solublemolecule, such as, a drug, forms a composite nanoparticle ornanoaggregate, wherein the drug is dispersed or deposited primarily atthe surface of the structures where hydrophobic portions or sites arelocated. The particles or aggregates of interest are stable, forexample, can be desiccated and rehydrated. The nanoparticles ornanoaggregates can range from about 50 nm to about 500 nm in diameterdepending, in part, on the drug to polymer ratio, the drug, the polymer,the solvent(s) used, amount of the homopolymer and amount of the drug.Hydrophobic, electrostatic, metal-ligand interactions, hydrogen bondingand other molecular interactions may be involved in the spontaneousinteractions between the water insoluble or poorly water solublemolecule and the homopolymer to form aggregates. The particles oraggregates of interest have a controlled release profile and thus findutility, for example, as a carrier for the controlled release ofpharmacologically active agents, drugs and the like in a host, forproviding a supplement, nutrient or requirement; for treating any of avariety of disorders; and the like.

BACKGROUND Symmetrically Branched Polymers

A new class of polymers called dendritic polymers, including Starburstdendrimers (or Dense Star polymers) and Combburst dendrigrafts (or hypercomb branched polymers), recently was developed and studied for variousindustrial applications. Those polymers often possess: (a) a welldefined core molecule, (b) at least two concentric dendritic layers(generations) with symmetrical (equal length) branches and branchjunctures, and (c) exterior surface groups, such as, polyamidoamine(PAMAM)-based branched polymers and dendrimers described in U.S. Pat.Nos. 4,435,548; 4,507,466; 4,568,737; 4,587,329; 5,338,532; 5,527,524;and 5,714,166. Other examples include polyethyleneimine (PEI)dendrimers, such as those disclosed in U.S. Pat. No. 4,631,337;polypropyleneimine (PPI) dendrimers, such as those disclosed in U.S.Pat. Nos. 5,530,092; 5,610,268; and 5,698,662; Frechet-type polyetherand polyester dendrimers, core shell tectodendrimers and others, asdescribed, for example, in “Dendritic Molecules”, edited by Newkome etal., VCH Weinheim, 1996; “Dendrimers and Other Dendritic Polymers”,edited by Frechet & Tomalia, John Wiley & Sons, Ltd., 2001; and U.S.Pat. No. 7,754,500.

Combburst dendrigrafts are constructed with a core molecule andconcentric layers with symmetrical branches through a stepwise syntheticmethod. In contrast to dendrimers, Combburst dendrigrafts or polymersare generated with monodisperse linear polymeric building blocks (U.S.Pat. Nos. 5,773,527; 5,631,329 and 5,919,442). Moreover, the branchpattern is different from that of dendrimers. For example, Combburstdendrigrafts form branch junctures along the polymeric backbones (chainbranches), while Starburst dendrimers often branch at the termini(terminal branches). Due to the living polymerization techniques used,the molecular weight distributions (M_(w)/M_(n)) of those polymers (coreand branches) often are narrow. Thus, Combburst dendrigrafts producedthrough a graft-on-graft process are well defined with M_(w)/M_(n)ratios often less than about 1.

SBP's, such as dendrimers, are predominantly produced by repetitiveprotecting and deprotecting procedures through either a divergent or aconvergent synthetic approach. Since dendrimers utilize small moleculesas building blocks for the cores and the branches, the molecular weightdistribution of the dendrimers often is defined. In the case of lowergenerations, a single molecular weight dendrimer often is obtained.

In addition to dendrimers and dendrigrafts, other SBP's includesymmetrical star shaped or comb shaped polymers, such as, symmetricalstar shaped or comb shaped polyethyleneoxide (PEO), polyethyleneglycol(PEG), PEI, PPI, polyoxazoline (POX), polymethyloxazoline (PMOX),polyethyloxazoline (PEOX), polystyrene, polymethylmethacrylate,polydimethylsiloxane or a combination thereof.

Asymmetrically Branched Polymers

Unlike SBP's, asymmetrically branched polymers (ABP), particularlyasymmetrically branched dendrimers or regular ABP (reg-ABP), oftenpossess a core, controlled and well defined asymmetrical (unequallength) branches and asymmetrical branch junctures as described in U.S.Pat. Nos. 4,289,872; 4,360,646; and 4,410,688.

On the other hand, a random ABP (ran-ABP) possesses: a) no core, b)functional groups both at the exterior and in the interior, c)random/variable branch lengths and patterns (i.e., termini and chainbranches), and d) unevenly distributed interior void spaces.

The synthesis and mechanisms of ran-ABPs, such as, made of PEI, wasreported by Jones et al., J. Org. Chem. 9, 125 (1944), Jones et al., J.Org. Chem. 30, 1994 (1965) and Dick et al., J. Macromol. Sci. Chem., A4(6), 1301-1314, (1970)). Ran-ABP, such as those made of POX, i.e.,poly(2-methyloxazoline) and poly(2-ethyloxazoline), were reported byLitt (J. Macromol. Sci. Chem. A9(5), 703-727 (1975)) and Warakomski (J.Polym. Sci. Polym. Chem. 28, 3551 (1990)). The synthesis of ran-ABP'soften can involve a one-pot divergent or a one-pot convergent method.

Homopolymers

A homopolymer can relate to a polymer or to a polymer backbone composedof the same repeat unit, that is, the hompolymer is generated from thesame monomer (e.g., polyethyleneimine dendrimers, polyamidoaminedendrimers or polyoxazoline dendrimers). The monomer can be a simplecompound or a complex or an assemblage of compounds where the assemblageor complex is the repeat unit in the homopolymer. Thus, if an assemblageis composed of three compounds, A, B and C; the complex can be depictedas ABC. A polymer composed of (ABC)-(ABC)-(ABC) . . . is a homopolymerfor the purposes of the instant disclosure. The homopolymer may belinear or branched. Thus, in the case of a randomly branched PEI,although there are branches of different length and branches occurrandomly, that molecule is a homopolymer for the purposes of the instantdisclosure because that branched polymer is composed of a singlemonomer, ethyleneimine or aziridine. Also, one or more of the monomer orcomplex monomer components can be modified, substituted, derivatized andso on, for example, modified to carry a functional group. Such moleculesare homopolymers for the purposes of the instant disclosure as thebackbone is composed of a single simple or complex monomer.

Poorly Water Soluble Drugs

Small molecule drug candidates and drugs, as well as biologicalmolecules, which can be modified for particular purposes or to haveparticular properties, may be poorly soluble or insoluble in water.Generally, the need for hydrophilicity for a molecule to survive incirculation or in tissue spaces can constrain the use ofpharmacologically active hydrophobic drug candidates or drugs. Hence,development of effective formulations for poorly water solublepharmaceutically active agents (PAA) is important in drug developmentand use. Current solutions include improving drug solubility or reducingdrug particle size by, for example, chemical modification or physicalformulation.

Chemical modification methods often involve converting the drug, e.g.,by using a salt form, hydrating or attaching various water solublefunctional groups, such as, amino/imino, hydroxyl, or carboxylcontaining groups; water soluble polymers, such as, PEG or PEO, and thelike to the original drug molecule to enhance water solubility.

Physical formulation can include using a cosolvent and/or a surfactantto dissolve a poorly soluble drug; involving a lipid or a liposome-basednanoemulsion or microemulsion; melting drug and polymer without anysolvents at elevated temperatures; using a complexing agent (e.g., aninorganic salt, coordination metals (e.g., hexamine cobalt (III)chloride), chelates (e.g., EDTA, EGTA etc.), metal-olefins ormetallocenes (e.g., Ferrocene), inclusion compounds (e.g.,cyclodextrins, choleic acid etc.) or molecular complexes); as well assolid dispersion in a carrier, such as, e.g., acids, such as, citricacid, tartaric acid, succinic acid, HCl etc.), sugars (e.g., dextrose,sorbitol, sucrose, maltose, galactose, xylitol etc.), polymericmaterials (e.g., polyvinylpyrrolidone, PEG-400, PEG-1000, PEG-4000,PEG-6000, carboxymethyl cellulose, hydroxypropyl cellulose, guar gums,xanthan gums, sodium alginates, methyl celluloses, HPMC, cyclodextrinsand their derivatives, galactomannans, surfactants (e.g.,polyoxyethylene stearate, a poloxamer, a deoxycholic acid, a Tween, aSpan, a Gelucire, a vitamin E TPGS etc.), and the like (e.g.,pentaerythritol, urea, urethane, hydroxyalkyl xanthenes etc.).

Other known strategies include drug particle size reduction, forexample, micronization, which can use a milling technique, such as, useof a jet mill or a rotor stator colloid mill to reduce particle size;increase dissolution rate with increased surface area; nanosuspension,which is a submicron colloidal dispersion of pure particles of drugs,which can be stabilized by surfactants; homogenization, which ofteninvolves conventional homogenizers, sonicators and high shear fluidprocessors; wet milling, where the active drug is fragmented in thepresence of surfactant by milling or by spraying drug dissolved in avolatile organic solvent into a heated aqueous solution; usingsupercritical fluids; polymorph changes; using eutectic mixtures; usingself microemulsifying drug delivery systems etc.

However, those treatments may compromise pharmacologic activity.

While drugs often can be delivered through various routes, includingoral, intrathecal, rectal, intranasal, subdermal, subdural,intramuscular, transdermal, topical, inhalation, injection and so on,intravenous drug delivery allows rapid and direct equilibration of thedrug in the circulation, that can enable effective local concentration.A stable and controlled drug release formulation not only can avoidexcessively high serum levels just after dosing but also can allowgradual release of the drug in the intravascular compartment.

Microparticles larger than 7 μm are generally cleared from thecirculation by the “blood filtering organs,” such as, the spleen, lungsand liver. Therefore, smaller nanoparticles, e.g., 50-500 nm, oftenpossess longer blood circulation times.

Examples of pharmaceutically active agents (PAA), such as, drugs,include, but are not limited to, chlormethine, chlorambucil, busulfan,thiotepa, cyclophosphamide, estramustine, ifosfamide, meclilorethamine,melphalan, uramustine, lonuistine, streptozotocin, dacarbazine,procarbazine, temozolainide, cisplatin, carboplatin, oxaliplatin,satraplatin, (SP-4-3)-(cis)-aminedichloro-[2-methylpyridine]-platinum(II), methotrexate, permetrexed, raltitrexed, trimetrexate,camptothecin, camptothecin derivatives (such as, irinotecan, topotecanetc.), cladribine, chlorodeoxyadenosine, clofarabine, fludarabine,mercaptopurine, pentostatin, thioguanine, azacitidine, capecitabine,cytarabine, edatrexate, floxuridine, 5-fluorouracil, gemcitabine,troxacitabine, bleomycin, dactinomycin, adriamycin, actinomycin,mithramycin, mitomycin, mitoxantrone, porfiromycin, daunorubicin,doxorubicin, liposomal doxorubicin, epirubicin, idarubicin, valrubicin,phenesterine, tamoxifen, piposulfancamptothesin, L-asparaginase,PEG-L-asparaginase, paclitaxel, docetaxel, taxotere, vinblastine,vincristine, vindesine, vinorelbine, irinotecan, topotecan, amsacrine,etoposide, teniposide, fluoxymesterone, testolactone, bicalutamide,cyproterone, flutamide, nilutamide, aminoglutethimide, anastrozole,exemestane, formestane, letrozole, dexamethasone, prednisone,diethylstilbestrol, fulvestrant, raloxifene, toremifene, buserelin,goserelin, leuprolide, triptorelin, medroxyprogesterone acetate,megestrol acetate, levothyroxine, liothyronine, altretamine, arsenictrioxide, gallium nitrate, hydroxyurea, levamisole, mitotane,octreotide, procarbazine, suramin, thalidomide, methoxsalen, sodiumporfimer, bortezomib, erlotinib hydrochloride, gefitinib, imatinibmesylate, semaxanib, adapalene, bexarotene, trans-retinoic acid,9-cis-retinoic acid and N-(4-hydroxyphenyl) retinamide, alemtuzumab,bevacizumab, cetuximab, ibritumomab tiuxetan, rituximab, trastuzumab,gemtuzumab ozogamicin, tositumomab, interferon-α2a, interferon-α and soon, and derivatives and modifications thereof, so long as the drug, orderivative thereof, is poorly soluble or insoluble in water. Some of themolecules above are modified to be more soluble in water. For thepurposes of the instant disclosure, such molecules can be modified oraltered to remove such modifications resulting in a pharmaceuticallyactive or biologically active molecule which is less hydrophilic andmore hydrophobic, that is, poorly water soluble or water insoluble.

Thus, PAA's that are water insoluble or poorly water soluble, or thosewhich are sensitive to acid environments generally cannot beconventionally administered (e.g., by intravenous injection or oraladministration). In some circumstances, parenteral administration ofsuch pharmaceuticals can be achieved by emulsification ofoil-solubilized drug with an aqueous liquid (such as normal saline),often in the presence of surfactants or emulsifiers to produce anemulsion for administration.

For example, paclitaxel is a water insoluble drug. Paclitaxel is sold asTaxol® by Bristol-Myers Squibb. Paclitaxel is derived from the PacificYew tree, Taxus brevifolia (Wan et al., J. Am. Chem. Soc. 93:2325(1971). Taxanes, including paclitaxel and docetaxel (also sold asTaxotere®) are used to treat various cancers, including, breast, ovarianand lung cancers, as well as colon, and head and neck cancers, etc.

However, the poor aqueous solubility of paclitaxel has hampered thewidespread use thereof. Currently, Taxol® and generics thereof areformulated using a 1:1 solution of ethanol:Cremaphor® (polyethyoxylatedcastor oil) to solubilize the drug. The presence of Cremaphor® has beenlinked to severe hypersensitivity reactions and consequently requiresmedication of patients with corticosteroids (e.g., dexamethasone) andantihistamines.

Alternatively, conjugated paclitaxel, for example, Abraxane®, which isproduced by mixing paclitaxel with human serum albumin, has eliminatedthe need for corticosteroids and antihistamine injections. However,Abraxane® generates undesirable side effects, such as, severecardiovascular events, including chest pain, cardiac arrest,supraventricular tachycardia, edema, thrombosis, pulmonarythromboembolism, pulmonary emboli, hypertension etc, which preventspatients with high cardiovascular risk from using the drug.

Delivery of Poorly Water Soluble Drugs with Surface Modified BranchedPolymers

Although branched polymers, including SBP's and ABP's, have been usedfor drug delivery, those attempts are primarily focused on the chemicalattachment of the drug to the polymer, or physical encapsulation of suchdrugs in the interior through unimolecular encapsulation (U.S. Pat. Nos.5,773,527; 5,631,329; 5,919,442; and 6,716,450).

For example, dendrimers and dendrigrafts are believed to physicallyentrap bioactive molecules using unimolecular encapsulation approaches,as described in U.S. Pat. Nos. 5,338,532; 5,527,524; and 5,714,166 fordense star polymers, and U.S. Pat. No. 5,919,442 for hyper comb branchedpolymers. Similarly, the unimolecular encapsulation of various drugsusing SBP's to form a, “dendrimer box,” was reported in Tomalia et al.,Angew. Chem. Int. Ed. Engl., 1990, 29, 138, and in “Dendrimers and OtherDendritic Polymers”, edited by Frechet & Tomalia, John Wiley & Sons,Ltd., 2001, 387-424.

Branched core shell polymers with a hydrophobic core and a hydrophilicshell may be used to entrap a poorly water soluble drug throughmolecular encapsulation. Randomly branched and hyperbranched core shellstructures with a hydrophilic core and a hydrophobic shell have alsobeen used to carry a drug through unimolecular encapsulation andpre-formed nanomicelles (U.S. Pat. No. 6,716,450 and Liu et al.,Biomaterials 2010, 10, 1334-1341). However, those unimolecular andpre-formed micelle structures are generated in the absence of a drug.

Block copolymers, such as miktoarm polymers (i.e., Y shape/AB₂ type starpolymers) and linear (A)-dendritic (B) block copolymers, were observedto form sterocomplexes with paclitaxel (Nederberg et al.,Biomacromolecules 2009, 10, 1460-1468 and Luo et al., Bioconjugate Chem.2010, 21, 1216). Those block copolymers closely resemble traditionallipid or AB-type linear block copolymers, which are well knownsurfactants used for the generation of micelles.

However, such branched block copolymers are difficult to make and thus,are not suitable for mass production.

There are no descriptions of modifying branched homopolymers, which onexposure to a poorly soluble or water insoluble drug, spontaneously formstable aggregates which are suitable for controlled drug delivery.

SUMMARY

In one aspect, the present disclosure is directed to use of modifiedbranched polymers (MBP) to increase the solubility of water insoluble orpoorly water soluble pharmaceutically active agents (PAA), such as,drugs. Such MBP's can include both symmetrically and asymmetricallybranched polymers.

In another aspect of the disclosure, the symmetrically branched polymer(SBP) has regular symmetrical branches within the polymer. In anotheraspect of the disclosure, the asymmetrically branched polymer (ABP) haseither random or regular, asymmetrical branches. The random ABP can alsohave a mixture of terminal and chain branching patterns.

In another aspect of the disclosure, both ABP's and SBP's can bemodified further with at least one molecule or group capable of formingadditional branches at a given time so that new material properties canbe achieved, wherein additional functional groups may be furtherattached. All of the modified polymers can be defined as modifiedsymmetrically or asymmetrically branched polymers.

In another aspect of the disclosure, the unmodified and modifiedbranched polymers either can be produced by a divergent or a convergentmethod, and either a stepwise or a one-step synthetic process can beused.

In another aspect of the disclosure, the SBP includes, but is notlimited to, polyamidoamine dendrimers; polyethyleneimine dendrimers;polypropyleneimine dendrimers; polyether dendrimers; polyesterdendrimers; comb branched/star branched polymers, such as,polyamidoamine, polyethyleneoxide (PEO), polyethyleneglycol (PEG),polymethyloxazoline, polyethyloxazoline, polymethylmethacrylate (PMA),polystyrene, polybutadiene, polyisoprene and polydimethylsiloxane; combbranched dendrigrafts, such as, polyethyloxazoline, polymethyloxazoline,polyethyleneimine, polyamidoamine; and so on.

In a further aspect of the disclosure, the SBP can have an interior voidspace, while the ABP can have unevenly distributed void spaces.

In another aspect of the disclosure, a hybrid branched polymercomprising the aforementioned SBP's, such as, dendrimers ordendrigrafts, and ABP's, such as, regular and randomly branchedpolymers, as well as star branched and comb branched polymers, orcombination thereof, can also be used for the generation of saiddrug-induced aggregates or nanoparticles of interest.

In another aspect of the disclosure, the branched polymers are modifiedwith functional groups, such as, but not limited to, NH₂, NHR, NR₂, NR₃⁺, COOR, COOH, COO⁻, OH, C(O)R, C(O)NH₂, C(O)NHR or C(O)NR₂, wherein Rcan be any aliphatic group, aromatic group or combination thereof; analiphatic group (e.g., a hydrocarbon chain), which can be branched, cancontain one or more double and/or triple bonds and/or may besubstituted; an aromatic group, which may contain a plurality of rings,which may be fused or separated, the rings may be of varying size and/ormay contain substituents; perfluorocarbon chains; saccharides and/orpolysaccharides, which may be of varying ring sizes, the rings maycontain a heteroatom, such as a sulfur or a nitrogen atom, may besubstituted, may contain more than one species of saccharide, may bebranched and/or may be substituted; polyethylene glycols; and the like.

The molecular weight of the MBP's can range from about 500 to over5,000,000; from about 500 to about 1,000,000; from about 1,000 to about500,000; or from about 2,000 to about 100,000.

In another aspect of the disclosure, the surface of the symmetricallyand asymmetrically polymers is modified so that the physical propertiesof the surface groups will be more compatible with a PAA of interest,thus making the PAA more miscible with the surface group region/domainof the MBP's.

In an embodiment, the modification of branched polymers is withhydrophobic functional groups, such as, aliphatic chains (e.g.,hydrocarbon chains comprising 1 to about 22 carbons, whether linear orbranched), aromatic structures (e.g. containing one or more aromaticrings, which may be fused) or combinations thereof.

In contrast to known drug carriers, the PAA's of the instant disclosureare not physically entrapped within said branched polymer structures.Instead, the PAA either can be located at or dispersed in thedomains/regions containing surface functional groups of each branchedpolymer.

The resulting structures of interest optionally can be preserved, forexample, by lyophilization or other form of desiccation, which mayfurther stabilize the structures of interest. Once redissolved in wateror a buffer, nanoparticles with sizes ranging from about 50 to about 500nm in diameter can be obtained.

The presence of multiple, often functionalized branches enables theformation of intramolecular and intermolecular crosslinks, which maystabilize the PAA-containing nanoparticles. On dilution, said physicalaggregate or nanoparticle deconstructs releasing drug at a controlledrate.

In another aspect of the disclosure, the branched polymer can comprisetargeting moieties/groups including, but not limited to, an antibody orantigen-binding portion thereof, antigen, cognate carbohydrates (e.g.,sialic acid), a cell surface receptor ligand, a moiety bound by a cellsurface receptor, a moiety that binds a cell surface saccharide, anextracellular matrix ligand, a cytosolic receptor ligand, a growthfactor, a cytokine, an incretin, a hormone, a lectin, a lectin ligand,such as, a galactose, a galactose derivative, an N-acetylgalactosamine,a mannose, a mannose derivative and the like, a vitamin, such as, afolate or a biotin, avidin, streptavidin, neutravidin, DNA, RNA etc.Such targeted nanoparticles release drug at the preferred treatmentlocations, and therefore, enhance local effective concentrations and canminimize undesired side effects.

In another aspect of the disclosure, a targeting moiety/group and afunctional group, including, hydrophobic, hydrophilic and/or ionicfunctional groups, are attached to the branched polymer prior to theformation of the composite nanoparticle for targeted drug delivery.

In another aspect of the disclosure, a diagnostic agent, such as, acontrast reagent, also can be carried by said structures of interest,for example, for in vivo imaging. In an embodiment, the diagnostic agentis one which is poorly soluble or insoluble in water, thereby negating aneed for a drug to form a structure of interest.

In some embodiments, an imaging agent is one comprising a metal or isparamagnetic, e.g., magnetic resonance imaging materials, which can bedeposited or entrapped within said branched polymer of saidnanocomposite-based particle.

In yet another aspect of the disclosure, the diagnostic materialcontaining nanoparticles can further comprise a targeting moiety/group,which allows such nanoparticle to target specific locations fordiagnosis.

In other embodiments, a structure of interest comprises a second or morePAA's. The second or more PAA's may or may not be poorly soluble orinsoluble in water.

In another aspect of disclosure, the nanoparticle can carry pluralsimilar PAA's or can carry PAA's of different function or activity, suchas, types of drugs to form a combination or cocktail therapy. Such drugsmay include, but are not limited to, small molecule drugs, inorganicdrugs and biological molecule-based drugs, such as peptides, proteins,antibodies or antigen-binding portions thereof, enzymes, vaccines and soon, for the treatment of various diseases or general use, such as, forcosmetics and over the counter products.

In another aspect of the disclosure, the nanoparticle-based drugformulations also can be used in drug discovery and development, wherevarious therapeutic formulations can be screened and tested rapidly.

Additional features and advantages of the present disclosure aredescribed in, and will be apparent from, the following DetailedDescription and the attached Figures.

BRIEF DESCRIPTION OF THE FIGURES

The following description of the figures and the respective drawings arenon-limiting examples that depict various embodiments that exemplify thepresent disclosure.

FIGS. 1A-D depict SBP's including a dendrimer (FIG. 1A), a star shapedpolymer (FIG. 1D), a dendrigraft (FIG. 1B) and a comb shaped polymer(FIG. 1C). All have a core, whether globular or linear.

FIGS. 2A and 2B depict chemical structures of symmetrically branched PPIdendrimers.

FIG. 3 depicts chemical modification reactions of symmetrically branchedPPI dendrimers. The numbers, 8, 16, 32 and so on indicate the number ofreactive groups at the surface of the dendrimer.

FIGS. 4A and 4B depict random (A) and regular (B) ABP's withasymmetrical branch junctures and patterns.

FIG. 5 depicts a chemical structure of a random asymmetrically branchedPEI homopolymer.

FIGS. 6A and 6B depict synthetic schemes. FIG. 6A presents chemicalmodification reactions of random asymmetrically branched PEIhomopolymers. FIG. 6B depicts a one-pot synthesis of hydrophobicallymodified, randomly branched poly(2-ethyloxazoline) with a primary aminogroup at the focal point of the polymer. The initiator/surface group (I)is the brominated hydrocarbon. The reaction opens the oxazoline ring.

FIGS. 7A and 7B illustrate a drug loaded in or at the surface domain orregion of the branched polymer (SBP's (FIG. 7A) and ABP's (FIG. 7B)). Inthis and other figures, R indicates a surface group and a solid circledepicts a drug of interest.

FIG. 8 illustrates one type of composite-based nanoparticles containingboth drug molecules and branched polymers.

FIGS. 9A and 9B an insoluble or poorly water soluble drug that is loadedat hydrophobic surface groups of branched polymers (SBP's (FIG. 9A) andABP's (FIG. 9B)). In this and other figures, a thin, wavy line depicts ahydrophobic surface group.

FIGS. 10A and 10B illustrate various drug-containing nanoparticles alsocarrying at least one targeting group or moiety, such as, an antibody,depicted herein and in other figures as a, “Y.” FIG. 10A depicts asymmetrically branched polymer and FIG. 10B depicts an asymmetricallybranched polymer.

FIGS. 11A and 11B illustrate drug-containing nanoparticles carrying bothmagnetic imaging contrast agents and a targeting moiety or group, suchas an antibody. In this and other Figures, M denotes an imagingmaterial, such as, a magnetic resonance imaging contrast agent. FIG. 11Adepicts a symmetrically branched polymer and FIG. 11B depicts anasymmetrically branched polymer.

FIGS. 12A and 12B illustrate drug-containing nanoparticles carrying bothradioactive (Rad) agents and a targeting moiety or group, such as, anantibody. FIG. 12A depicts a symmetrically branched polymer and FIG. 12Bdepicts an asymmetrically branched polymer.

FIG. 13 shows the size comparison of polymer-only and polymer-drugaggregates with the polymer concentration at 25 mg/mL and the drugconcentration at 5 mg/mL in saline. The polymer is ahydrophobically-modified, randomly-branched PEOX and the drug ispaclitaxel.

FIG. 14 shows the size comparison of polymer-only and polymer-drugaggregates with the polymer concentration at 2.5 mg/mL and the drugconcentration at 0.5 mg/mL in saline. The polymer is ahydrophobically-modified, randomly-branched PEOX and the drug ispaclitaxel.

FIG. 15 shows the size comparison of polymer-only and polymer-drugaggregates with the polymer concentration at 250 μg/mL and the drugconcentration at 50 μg/mL in saline. The polymer is ahydrophobically-modified, randomly-branched PEOX and the drug ispaclitaxel.

FIG. 16 shows the size comparison of polymer-only and polymer-drugaggregates with the polymer concentration at 25 μg/mL and the drugconcentration at 5 μg/mL in saline. The polymer is ahydrophobically-modified, randomly-branched PEOX and the drug ispaclitaxel.

FIG. 17 summarizes data comparing cytotoxicity of a drug and a drugaggregate. MCF-7 human breast cancer cells were exposed to variousconcentrations of each and survivability was determined.

FIG. 18 summarizes data comparing cytotoxicity of a drug and a drugaggregate. H460 human epithelial lung cancer cells were exposed tovarious concentrations of each and survivability was determined.

DETAILED DESCRIPTION OF THE DISCLOSURE

The drug solubility in this disclosure is defined as, relative to partsof solvent required to solubilize for one part of drug, <30 (soluble),30-100 (poorly soluble) and >100 (insoluble).

For the purposes of the instant disclosure, the words, such as, “about,”“substantially” and the like are defined as a range of values no greaterthan 20% from the stated value or figure. “Homopolymer,” is as describedhereinabove.

Drugs of Interest

The PAA described in this disclosure includes any chemical/smallmolecule-based drug, inorganic-based drug, biological/largemolecule-based drug, modifications and/or derivatives thereof, andcombinations thereof, wherein the drug is poorly soluble or insoluble inwater. Hence, a drug of interest can be a small molecule, a salt thereofin which the molecule is modified to be water insoluble or poorly watersoluble or can be a biological molecule which is modified to be waterinsoluble or poorly water soluble.

Chemical/small molecule drugs can include any substantially poorlysoluble or water insoluble pharmacologically active agents contemplatedfor use in the practice of the present disclosure include PAA's, drugs,imaging agents, diagnostic agents, agents of nutritional value,supplements, vitamins, lifestyle chemicals and the like. Some of thosemay need to be converted to a less water soluble form, for example,changing the PAA from a salt to a non-salt form or from a charged to anon-charged molecule.

Suitable examples of PAA's include drugs which are poorly soluble orinsoluble in water, which include, growth agents, AIDS adjunct agents,alcohol abuse preparations, such as, agents for treating dependence orwithdrawal, Alzheimer's Disease treatments, Amyotrophic LateralSclerosis treatments, analgesics, anesthetics, anticonvulsants,antidiabetic agents, antidotes, antifibrosis therapies, antihistamines,anti-infective agents, such as, antibiotics, antivirals, antifungals,amebicides, antihelmintics, antimalarials, leprostatics and so on,antineoplastics, antiparkinsonian agents, antirheumatic agents, appetitestimulants, biological response modifiers, biologicals, blood modifiers,such as, anticoagulants, colony stimulating factors, hemostatics, plasmaextenders, thrombin inhibitors and so on, bone metabolism regulators,cardioprotective agents, cardiovascular agents, such as, adrenergicblockers, adrenergic stimulators, ACE inhibitors, antiarrhythmics,antilipemic agents, calcium channel blockers, diuretics, vasopressorsand so on, CNS stimulants, cholinesterase inhibitors, contraceptives,fertility treatments, ovulation stimulators, cystic fibrosis managementsagents, detoxifying agents, diagnostics, dietary supplements, dopaminereceptor agonists, endometriosis management agents, enzymes, erectiledysfunction treatments, foot care products, GI agents, such as antacids,antidiarrheals, antiemetics, antiflatulants, bowel evacuants, digestiveenzymes, histamine receptor agonists, laxatives, proton pump inhibitors,prostaglandins and so on, Gaucher's Disease treatments, gout treatments,homeopathic remedies, skin treatments, vitamins, nutrients, hormones,hypercalcemia management treatments, hypocalcemia management treatments,immunomodulators, immunosuppressants, levocarnitine deficiencytreatments, mast cell stabilizers, migraine treatments, motion sicknessproducts, such as, benadryl and phenergan, decongestants,antihistamines, cough suppressants, multiple sclerosis treatments,muscle relaxants, nasal preparations, such as, antiinflammatories,smoking cessation aids, appetite suppressants, nucleoside analogs,obesity managements, ophthalmic preparations, such as, antibiotics,antiglaucoma agents, artificial tears, lubricants and so on, sexualaids, lubricants, osteoporosis treatments, otic preparations, such as,antiinfectives and cerumenolytics, minerals, oxytocics,parasympatholytics, parasympathomimetics, patent ductus arteriosusagents, phosphate binders, porphyria agents, prostaglandins,psychotherapeutic agents, radiopaque agents, respiratory agents, suchas, antiinflammatories, antitussives, bronchodilators, decongestants,expectorants, leukotrienes antagonists, surfactants and so on, saltsubstitutes, sedatives, hypnotics, skin and mucous membranepreparations, such as, acne treatments, anorectal treatments, such as,hemorrhoid treatments and enemas, antiperspirants, antipruritics,antipsoriatic agents, antiseborrheic agents, burn treatments, cleansingagents, depigmenting agents, emollients, hair growth retardants, hairgrowth stimulators, keratolytics, hair problem treatments, mouth andthroat problem treatments, shampoos, photosensitizing agents, warttreatments, wound care treatments and so on, over the counterpharmaceutics and products, such as, deodorants, Tourette's Syndromeagents, tremor treatments, urinary tract agents, such as, acidifiers,alkalinizers, antispasmodics, benign prostatic hyperplasia treatments,calcium oxalate stone preventors, enuresis management agents and so on,vaginal preparations, such as, antiinfectives, hormones and so on,vasodilators, vertigo treatments, Wilson's Disease treatments and so on.

Listed herein are drugs of interest as well as forms of drugs which maybe modified, for example, as salts. For the purposes of the invention,any such ionized or hydrophilic forms are modified as known in the artto remove such functional groups, modifications and the like to yieldnon-modified or other forms of drugs which are poorly soluble or notsoluble in water. Examples of pharmaceutically active agents, drugs andthe like include those listed herein and, for example,analgesics/antipyretics (e.g., aspirin, acetaminophen, ibuprofen,naproxen sodium, buprenorphine hydrochloride, propoxyphenehydrochloride, propoxyphene napsylate, meperidine hydrochloride,hydromorphone hydrochloride, morphine sulfate, oxycodone hydrochloride,codeine phosphate, dihydrocodeine bitartrate, pentazocine hydrochloride,hydrocodone bitartrate, levorphanol tartrate, diflunisal, trolaminesalicylate, nalbuphine hydrochloride, mefenamic acid, butorphanoltartrate, choline salicylate, butalbital, phenyltoloxamine citrate,diphenhydramine citrate, methotrimeprazine, cinnamedrine hydrochloride,meprobamate and the like); anesthetics (e.g., cyclopropane, enflurane,halothane, isoflurane, methoxyflurane, nitrous oxide, propofol and thelike); antiasthmatics (e.g., azelastine, ketotifen, traxanox, amlexanox,cromolyn, ibudilast, montelukast, nedocromil, oxatomide, pranlukast,seratrodast, suplatast tosylate, tiaramide, zafirlukast, zileuton,beclomethasone, budesonide, dexamethasone, flunisolide, triamcinoloneacetonide and the like); antibiotics (e.g., neomycin, streptomycin,chloramphenicol, cephalosporin, ampicillin, penicillin, tetracycline andthe like); antidepressants (e.g., nefopam, oxypertine, doxepinhydrochloride, amoxapine, trazodone hydrochloride, amitriptylinehydrochloride, maprotiline hydrochloride, phenelzine sulfate,desipramine hydrochloride, nortriptyline hydrochloride, tranylcyprominesulfate, fluoxetine hydrochloride, doxepin hydrochloride, imipraminehydrochloride, imipramine pamoate, nortriptyline, amitriptylinehydrochloride, isocarboxazid, trimipramine maleate, protriptylinehydrochloride and the like); antidiabetics (e.g., biguanides, hormones,sulfonylurea derivatives, and the like); antifungal agents (e.g.,griseofulvin, ketoconazole, amphotericin B, nystatin, candicidin and thelike); antihypertensive agents (e.g., propanolol, propafenone,oxyprenolol, nifedipine, reserpine, trimethaphan camsylate,phenoxybenzamine hydrochloride, pargyline hydrochloride, deserpidine,diazoxide, guanethidine monosulfate, minoxidil, rescinnamine, sodiumnitroprusside, rauwolfia serpentina, alseroxylon, phentolamine mesylate,reserpine and the like); anti-inflammatories (e.g., non-steroidalcompounds, such as, indomethacin, naproxen, ibuprofen, ramifenazone,piroxicam and so on, and steroidal compounds, such as, cortisone,dexamethasone, fluazacort, hydrocortisone, prednisolone, prednisone andthe like); antineoplastics (e.g., adriamycin, cyclophosphamide,actinomycin, bleomycin, daunorubicin, doxorubicin, epirubicin,mitomycin, methotrexate, fluorouracil, carboplatin, carmustine (BCNU),methyl-CCNU, cisplatin, etoposide, interferons, camptothecin andderivatives thereof, phenesterine, Taxol and derivatives thereof,taxotere and derivatives thereof, vinblastine, vincristine, tamoxifen,etoposide, piposulfan and the like); antianxiety agents (e.g.,lorazepam, buspirone hydrochloride, prazepam, chlordiazepoxidehydrochloride, oxazepam, clorazepate dipotassium, diazepam, hydroxyzinepamoate, hydroxyzine hydrochloride, alprazolam, droperidol, halazepam,chlormezanone, dantrolene and the like); immunosuppressive agents (e.g.,cyclosporine, azathioprine, mizoribine, FK506 (tacrolimus) and thelike); antimigraine agents (e.g., ergotamine tartrate, propanololhydrochloride, isometheptene mucate, dichloralphenazone and the like);sedatives/hypnotics (e.g., barbiturates (e.g., pentobarbital,pentobarbital sodium, secobarbital sodium and the like) orbenzodiazapines (e.g., flurazepam hydrochloride, triazolam, tomazeparm,midazolam hydrochloride and the like); antianginal agents (e.g.,β-adrenergic blockers, calcium channel blockers (e.g., nifedipine,diltiazem hydrochloride and the like) and nitrates (e.g., nitroglycerin,isosorbide dinitrate, pentaerythritol tetranitrate, erythrityltetranitrate and the like)); antipsychotic agents (e.g., haloperidol,loxapine succinate, loxapine hydrochloride, thioridazine, thioridazinehydrochloride, thiothixene, fluphenazine hydrochloride, fluphenazinedecanoate, fluphenazine enanthate, trifluoperazine hydrochloride,chlorpromazine hydrochloride, perphenazine, lithium citrate,prochlorperazine and the like); antimanic agents (e.g., lithiumcarbonate); antiarrhythmics (e.g., bretylium tosylate, esmololhydrochloride, verapamil hydrochloride, amiodarone, encainidehydrochloride, digoxin, digitoxin, mexiletine hydrochloride,disopyramide phosphate, procainamide hydrochloride, quinidine sulfate,quinidine gluconate, quinidine polygalacturonate, flecainide acetate,tocainide hydrochloride, lidocaine hydrochloride and the like);antiarthritic agents (e.g., phenylbutazone, sulindac, penicillamine,salsalate, piroxicam, azathioprine, indomethacin, meclofenamate sodium,gold sodium thiomalate, ketoprofen, auranofin, aurothioglucose, tolmetinsodium and the like); antigout agents (e.g., colchicine, allopurinol andthe like); anticoagulants (e.g., heparin, heparin sodium, warfarinsodium and the like); thrombolytic agents (e.g., urokinase,streptokinase, altoplase and the like); antifibrinolytic agents (e.g.,aminocaproic acid); hemorheologic agents (e.g., pentoxifylline);antiplatelet agents (e.g., aspirin, empirin, ascriptin and the like);anticonvulsants (e.g., valproic acid, divalproate sodium, phenytoin,phenytoin sodium, clonazepam, primidone, phenobarbitol, phenobarbitolsodium, carbamazepine, amobarbital sodium, methsuximide, metharbital,mephobarbital, mephenytoin, phensuximide, paramethadione, ethotoin,phenacemide, secobarbitol sodium, clorazepate dipotassium, trimethadioneand the like); antiparkinson agents (e.g., ethosuximide and the like);antihistamines/antipruritics (e.g., hydroxyzine hydrochloride,diphenhydramine hydrochloride, chlorpheniramine maleate, brompheniraminemaleate, cyproheptadine hydrochloride, terfenadine, clemastine fumarate,triprolidine hydrochloride, carbinoxamine maleate, diphenylpyralinehydrochloride, phenindamine tartrate, azatadine maleate, tripelennaminehydrochloride, dexchlorpheniramine maleate, methdilazine hydrochloride,trimprazine tartrate and the like); agents useful for calcium regulation(e.g., calcitonin, parathyroid hormone and the like); antibacterialagents (e.g., amikacin sulfate, aztreonam, chloramphenicol,chloramphenicol palmitate, chloramphenicol sodium succinate,ciprofloxacin hydrochloride, clindamycin hydrochloride, clindamycinpalmitate, clindamycin phosphate, metronidazole, metronidazolehydrochloride, gentamicin sulfate, lincomycin hydrochloride, tobramycinsulfate, vancomycin hydrochloride, polymyxin B sulfate, colistimethatesodium, colistin sulfate and the like); antiviral agents (e.g.,interferon γ, zidovudine, amantadine hydrochloride, ribavirin, acyclovirand the like); antimicrobials (e.g., cephalosporins (e.g., cefazolinsodium, cephradine, cefaclor, cephapirin sodium, ceftizoxime sodium,cefoperazone sodium, cefotetan disodium, cefutoxime azotil, cefotaximesodium, cefadroxil monohydrate, ceftazidime, cephalexin, cephalothinsodium, cephalexin hydrochloride monohydrate, cefamandole nafate,cefoxitin sodium, cefonicid sodium, ceforanide, ceftriaxone sodium,ceftazidime, cefadroxil, cephradine, cefuroxime sodium and the like),penicillins (e.g., ampicillin, amoxicillin, penicillin G benzathine,cyclacillin, ampicillin sodium, penicillin G K, penicillin V K,piperacillin sodium, oxacillin sodium, bacampicillin hydrochloride,cloxacillin sodium, ticarcillin disodium, azlocillin sodium,carbenicillin indanyl sodium, penicillin G procaine, methicillin sodium,nafcillin sodium and the like), erythromycins (e.g., erythromycinethylsuccinate, erythromycin, erythromycin estolate, erythromycinlactobionate, erythromycin stearate, erythromycin ethylsuccinate and thelike), tetracyclines (e.g., tetracycline hydrochloride, doxycyclinehyclate, minocycline hydrochloride and the like), and the like);anti-infectives (e.g., GM-CSF); bronchodilators (e.g., sympathomimetics(e.g., epinephrine hydrochloride, metaproterenol sulfate, terbutalinesulfate, isoetharine, isoetharine mesylate, isoetharine hydrochloride,albuterol sulfate, albuterol, bitolterol, mesylate isoproterenolhydrochloride, terbutaline sulfate, epinephrine bitartrate,metaproterenol sulfate, epinephrine, epinephrine bitartrate);anticholinergic agents (e.g., ipratropium bromide); xanthines (e.g.,aminophylline, dyphylline, metaproterenol sulfate, aminophylline); mastcell stabilizers (e.g., cromolyn sodium); inhalant corticosteroids(e.g., flunisolide, beclomethasone dipropionate monohydrate and thelike), salbutamol, beclomethasone dipropionate (BDP), ipratropiumbromide, budesonide, ketotifen, salmeterol, xinafoate, terbutalinesulfate, triamcinolone, theophylline, nedocromil sodium, metaproterenolsulfate, albuterol, flunisolide and the like); hormones (e.g., androgens(e.g., danazol, testosterone cypionate, fluoxymesterone,ethyltostosterone, testosterone enanthate, methyltestosterone,fluoxymesterone, testosterone cypionate and the like); estrogens (e.g.,estradiol, estropipate, conjugated estrogens and the like), progestins(e.g., methoxyprogesterone acetate, norethindrone acetate and the like),corticosteroids (e.g., triamcinolone, betamethasone, betamethasonesodium phosphate, dexamethasone, dexamethasone sodium phosphate,dexamethasone acetate, prednisone, methylprednisolone acetatesuspension, triamcinolone acetonide, methylprednisolone, prednisolonesodium phosphate methylprednisolone sodium succinate, hydrocortisonesodium succinate, methylprednisolone sodium succinate, triamcinolonehexacatonide, hydrocortisone, hydrocortisone cypionate, prednisolone,fluorocortisone acetate, paramethasone acetate, prednisolone tebulate,prednisolone acetate, prednisolone sodium phosphate, hydrocortisonesodium succinate and the like), thyroid hormones (e.g., levothyroxinesodium); and the like); and the like; hypoglycemic agents (e.g., humaninsulin, purified beef insulin, purified pork insulin, glyburide,chlorpropamide, glipizide, tolbutamide, tolazamide and the like);hypolipidemic agents (e.g., clofibrate, dextrothyroxine sodium,probucol, lovastatin, niacin and the like); proteins (e.g., DNase,alginase, superoxide dismutase, lipase and the like); nucleic acids(e.g., sense or anti-sense nucleic acids encoding any therapeuticallyuseful protein, including any of the proteins described herein and thelike); agents useful for erythropoiesis (e.g., erythropoietin);antiulcer or antireflux agents (e.g., famotidine, cimetidine, ranitidinehydrochloride and the like); antinauseants or antiemetics (e.g.,meclizine hydrochloride, nabilone, prochlorperazine, dimenhydrinate,promethazine hydrochloride, thiethylperazine, scopolamine and the like);oil-soluble vitamins (e.g., vitamins A, D, E, K and the like); and aswell as other drugs such as mitotane, visadine, halonitrosoureas,anthrocyclines, ellipticine and the like.

Examples of diagnostic agents contemplated for use in the practice ofthe present disclosure include, but are not limited to, for example,magnetic resonance imaging contrast agents (e.g., various metal ions,such as, gadolinium based compounds for functional MRI, fluorocarbons,lipid soluble paramagnetic compounds and the like), ultrasound contrastagents, radiocontrast agents, such as, conventional radionuclides, suchas, iodine, copper, fluorine, gallium, thallium and the like, which maybe complexed with a carrier (e.g., iodo-octanes, halocarbons, renografinand the like), as well as other diagnostic agents which cannot readilybe delivered without some physical and/or chemical modification toaccommodate the substantially water insoluble nature thereof. Metals andradionuclides can be carried or bound to a protein, lipid, nucleic acid,chelator, such as a small molecule, or combinations thereof.

Examples of agents of nutritional or lifestyle value contemplated foruse in the practice of the present disclosure include amino acids,sugars, lipids, proteins, carbohydrates, oils, such as, fish oil,fat-soluble vitamins (e.g., vitamins A, D, E, K and the like), minerals,supplements, fats, emollients, tanning agents, moisturizers and thelike, or combinations thereof.

Nanocomposite or Nanoaggregate

A nanocomposite is a physical mixture of two or more materials orcomponents (e.g., polymer and PAA molecules). In the instant disclosure,such a mixture could contain different nanoscopic phases or domainsformed between the PAA and branched homopolymer molecule in either solidor liquid states. Nanocomposite can include a combination of a bulkmatrix (e.g., branched homopolymers and PAA's) and nanodimensionalphase(s), which may exhibit different properties due to dissimilaritiesof structure and chemistry (e.g., the domain formed by the PAA and thesurface groups of branched polymer, as well as the domains formed by theinterior of the branched polymers). Since the solubility of thedomains/phases may be different, on dissolving the nanocomposite in anaqueous solution, one of the phases may dissolve faster than the otheror others, resulting in a gradual breakdown of the composite aggregateresulting in a graded and controlled release of the composite componentsand optionally, reformation of one or more of the components into anovel form, such as, a new aggregate.

The size of the aggregates described in the disclosure ranges frombetween about 10 to about 500 nm in diameter, or from about 30 nm toabout 300 nm in diameter. Aggregates may exhibit size-related propertiesthat differ significantly from those observed for microparticles or bulkmaterials.

SBP's are depicted in FIG. 1, with symmetric branches, wherein all thehomopolymers of interest possess a core and exhibit symmetric branchjunctures consisting either of terminal or chain branches throughout thehomopolymer. The functional groups are present predominantly at theexterior.

The modified SBP's can be obtained, for example, through chemicallylinking functional groups on, for example, symmetrically branched PAMAMor PPI dendrimers, commercially available from Aldrich, polyetherdendrimers, polyester dendrimers, comb branched/star branched polymers,such as, those containing PEO, PEG, PMOX or PEOX, polystyrene, and combbranched dendrigrafts, such as, those containing PEOX, PMOX or PEI.

The synthetic procedures for making such SBP's/dendrimers are known(see, for example, “Dendrimers and Other Dendritic Polymers,” Frechet &Tomalia, eds., John Wiley & Sons, Ltd., 2001) using commerciallyavailable reagents (for example, various generations of PPI dendrimers,FIG. 2) or a number of SBP's are commercially available. The synthesisof comb branched and combburst polymers is known (see, for example, U.S.Pat. Nos. 5,773,527; 5,631,329; and 5,919,442).

The higher branching densities of SBP's render the polymers molecularlycompact with a well defined interior void space, which makes suchmolecules suitable as a carrier for entities, such as, reporters orPAA's entrapped or encased therein.

The surface modifications can enhance the properties and uses of theresulting modified SBP's. For example, with suitable modification, awater insoluble SBP can become water soluble, while an SBP with a highcharge density can be modified to carry very low or no charge on thepolymer or at the polymer surface. On the other hand, a water solubleSBP can be modified with hydrophobic surface groups to enhance theability to solubilize water insoluble or poorly water soluble drugs atthe surface thereof.

In one embodiment of the instant disclosure, the SBP (for example,either a symmetrically branched PEI dendrimer, a PPI dendrimer, a PAMAMdendrimer or a symmetrically branched PEI dendrigraft) can be modifiedwith different kinds of, for example, primary amine groups through, forexample, Michael addition or an addition of acrylic esters onto aminegroups of the homopolymer. Thus, for example, through a Michael additionreaction, methyl acrylate can be introduced onto the primary and/orsecondary amino groups of PEI, PPI and polylysine (PLL) homopolymers.The ester groups then can be further derivatized, for example, by anamidation reaction. Thus, for example, such an amidation reaction with,for example, ethylenediamine, can yield the addition of an amino groupat the terminus of the newly formed branch. Other modifications to thehomopolymer can be made using known chemistries, for example, asprovided in, “Poly(amines) and Poly(ammonium salts)” in Handbook ofPolymer Synthesis (Part A), Kricheldorf, ed., New York, Marcel Dekker,1994; and “Dendrimers and Other Dendritic Polymers,” Frechet & Tomalia,eds., John Wiley & Sons, Ltd., 2001.

On such addition, a modified SBP, such as, a modified PEI, PPI, PAMAMdendrimer or PEI dendrigraft, is formed. As an extension of the SBP,such as PPI and PEI, the resulting modified SBP also is symmetricallybranched. Depending on the solvent environment (i.e. pH or polarity),the surface functional groups can carry different charge and/or chargedensity, and/or hydrophobic groups. The molecular shape and surfacefunctional group locations (i.e., surface functional group back folding)then can be tuned further, based on those characteristic properties.

In another embodiment of the disclosure, the modified SBP's can beproduced using any of a variety of synthetic schemes that, for example,are known to be amenable to reaction with a suitable site on thehomopolymer. Moreover, any of a variety of reagents can be used in asynthetic scheme of choice to yield any of a variety of modifications oradditions to the homopolymer backbone. Thus, for example, in the case ofthe Michael addition reaction to an amine described above, the additionof any of a variety of substituents can be used, for example, at thealkylation stage, using for example, any of a variety of acrylatereagents, such as, an acrylate comprising a hydrocarbon substituent,such as saturated or unsaturated hydrocarbons comprising 1 to about 22carbons, which my be substituted, aliphatic, aromatic, ringed, saturatedat one or more bonds or a combination thereof. Thus, suitable reactantsinclude, methyl acrylate, ethyl acrylate, propyl acrylate, butylacrylate, pentyl acrylate, hexyl acrylate, heptyl acrylate, octylacrylate, nonyl acrylate, decyl acrylate, undecyl acrylate, dodecylacrylate and so on, and mixtures thereof. Similarly, at the amidationstage in the example exemplified above, any of a variety of amines canbe used. For example, ethylenediamine, monoethanolamine,tris(hydroxymethyl)aminomethane, alkyl amine, allyl amine, or any aminomodified polymer, including those comprising PEG, PEO,perfluoropolymers, polystyrene, polyethylene, polydimethylsiloxane,polyacrylate, polymethylmethacrylate and the like, and mixtures thereof,can be used.

Such a synthetic strategy would allow not only symmetric growth of themolecule, where more branches with different chemical compositions canbe introduced, but also the addition of multiple functional groups atthe exterior of the structure. The precursor homopolymer can bemodified, and continuously, using the same or a different syntheticprocess until the desired SBP's with appropriate molecular weight andfunctional groups are attained. In addition, the hydrophobic andhydrophilic properties, as well as charge densities of such polymers,can be tailored to fit specific application needs using appropriatemonomers for constructing the homopolymer and suitable modificationreactions.

In another embodiment of the disclosure, if a divergent syntheticprocedure is used, the chain end of symmetrically star branched or combbranched homopolymer, such as, a poly(2-substituted oxazoline),including, for example, poly(2-methyloxazoline), poly(2-ethyloxazoline),poly(2-propyloxazoline) and poly(2-butyloxazoline, etc.), PEI,PEO/glycol, polyvinylpyrrolidone, polyphosphate, polyvinyl alcohol orpolystyrene, can be modified with another small molecule or polymer togenerate various functional groups at the homopolymeric chain endsincluding a primary, secondary or tertiary amine, carboxylate, hydroxyl,aliphatic (e.g., hydrocarbon chain), aromatic, fluoroalkyl, aryl, PEG,PEO, acetate, amide and/or ester groups. Alternatively, variousinitiators also can be utilized so that the same type of functionalgroups can be introduced at the chain end if a convergent syntheticapproach is utilized (Dendritic Molecules, Newkome et al., eds., VCH,Weinheim, 1996; Dendrimers and Other Dendritic Polymers, Frechet &Tomalia, eds., John Wiley & Sons, Ltd., 2001; and J. Macromol. Sci.Chem. A9(5), pp. 703-727 (1975)).

ABP's are depicted in FIGS. 4A and 4B with asymmetric branches, whereinsome of the polymers of interest possess no core and exhibitasymmetrical branch junctures consisting of both chain and terminalbranches throughout the entire homopolymer. The functional groups oftenare present both at the exterior and in the interior. However, when alarger functional group (e.g., a large hydrophobic or hydrophilic group)is used, the functional groups often can be attached preferentially andperhaps necessarily at the exterior of the ABP, for example, possiblydue to steric effects. Therefore, such surface MBP's can be utilized forsolubilization of or aggregate formation with an insoluble or poorlysoluble drug.

The modified ABP's can be obtained, for example, through chemicallylinking functional groups on regular ABP's, such as, polylysine (e.g.,branched PLL), on random ABP's, such as, PEI's (commercially availablefrom Aldrich, Polysciences, or BASF under the trade name, Luposal™) orpolyoxazolines, which can be prepared according to the procedure of Litt(J. Macromol. Sci. Chem. A9(5), pp. 703-727 (1975)). Other ABP's caninclude, but are not limited to, polyacrylamides, polyphosphates,polyvinylpyrrolidones, polyvinyl alcohols, etc.

A variety of known starting materials can be used. For making suchmodified ABP's. Such monomers and polymers are available commercially inlarge quantities at modest cost. For example, one such precursor monomerthat can be used to synthesize a homopolymer of interest is PEI. Thesynthesis of random asymmetrically branched PEI's is known (Jones etal., J. Org. Chem. 9, 125 (1944)). PEI's with various molecular weightsare available commercially from different sources, such as, Aldrich,Polysciences and BASF (under the trade name Luposal™). The randomasymmetrically branched PEI's are produced primarily through cationicring opening polymerization of ring strained cyclic imine monomers, suchas aziridines (ethyleneimine) and azetidines (propyleneimine), withLewis or Bronsted acids as initiators. (Dermer et al., “Ethylenediamineand Other Aziridines”, Academic Press, New York, (1969); and Pell, J.Chem. Soc. 71 (1959)). Since many of the methods are essentially one-potprocesses, large quantities of random ABP's can be readily produced.Randomly branched poly(2-substituted oxazoline) polymers can be preparedusing the procedure of Litt (J. Macromol. Sci. Chem. A9 (5), pp. 703-727(1975)).

The synthetic processes for making ABP's often generate various branchjunctures within the macromolecule. In other words, a mixture ofterminal and chain branch junctures is distributed throughout themolecular structure. The branching densities of the random ABP's can belower, and the molecular structure can be more open when compared withdendrimers and dendrigrafts. Although the branch pattern is random, theaverage ratio of primary, secondary and tertiary amine groups can berelatively consistent with a ratio of about 1:2:1, as described by Dicket al., J. Macromol. Sci. Chem., A4 (6), 1301-1314 (1970) and Lukovkin,Eur. Polym. J. 9, 559 (1973).

The presence of the branch junctures can make the random ABP's, such as,asymmetrically branched PEI's, form macromolecules with a possiblespherical, ovoid or similar configuration. Within the globularstructure, there are various sizes of pockets formed from the imperfectbranch junctures at the interior of the macromolecule. Unlike dendrimersand dendrigrafts where interior pockets are always located around thecenter core of the molecule, the pockets of random ABP's are spreadunevenly throughout the entire molecule. As a result, random ABP'spossess both exterior and unevenly distributed interior functionalgroups that can be further reacted with a variety of molecules, thusforming new macromolecular architectures, a modified random ABP ofinterest.

Although having a core, the functional groups of the regular ABP arealso distributed both at the exterior and in the interior, which is verysimilar to the random ABP. One such homopolymer is PLL, which can bemade as described in U.S. Pat. Nos. 4,289,872; 4,360,646; and 4,410,688.Such homopolymers also can be modified in a manner similar as that forrandom ABPs, as taught herein, and as known in the art.

In one embodiment of the disclosure, the ABP (for example, either arandom asymmetrically branched PEI or a regular asymmetrically branchedPLL) is modified with different kinds of primary amine groups through,for example, Michael addition or an addition of acrylic esters ontoamines of the polymer. Thus, for example, through a Michael additionreaction, methyl acrylate, or other acrylates as provided herein, can beintroduced onto the primary and/or secondary amino groups of, forexample, PEI and PLL homopolymers. The ester groups then can be furtherderivatized, for example, by an amidation reaction. Thus, for example,such an amidation reaction with, for example, ethylenediamine, can yieldthe addition of an amino group at the terminus of the newly formedbranch. Other modifications to the polymer can be made using knownchemistries, for example, as provided in “Poly(amines) and Poly(ammoniumsalts)” in “Handbook of Polymer Synthesis (Part A),” Kricheldorf, ed.,New York, Marcel Dekker, 1994.

On such addition, a modified ABP, such as, a modified PEI or PLLhomopolymer, is formed. As an extension of the ABP, such as PEI and PLL,the resulting modified ABP is also asymmetrically branched. Depending onthe solvent environment (i.e. pH or polarity), the surface functionalgroups can carry different charge and charge density. The molecularshape and functional group locations (i.e., functional group backfolding) then can be further tuned, based on those characteristicproperties.

In another embodiment, the modified ABP's can be produced using any of avariety of synthetic schemes that, for example, are known to be amenableto reaction with a suitable site on the homopolymer. Moreover, any of avariety of reagents can be used in a synthetic scheme of choice to yieldany of a variety of modifications or additions to the polymer backbone.Thus, for example, in the case of the Michael addition reaction to anamine described above, the addition of any of a variety of substituentscan be used at the alkylation stage, as provided hereinabove, forexample, with an acrylate, which can comprise a saturated or unsaturatedhydrocarbon, such as one comprising one carbon to about 22 carbons,which may be aliphatic, branched, saturated, aromatic, ringed orcombination thereof. Suitable reactants include methyl acrylate, ethylacrylate, propyl acrylate, butyl acrylate, pentyl acrylate, hexylacrylate, heptyl acrylate, octyl acrylate, nonyl acrylate, decylacrylate, undecyl acrylate, dodecyl acrylate and the like, and mixturesthereof. Similarly, at the amidation stage in the example exemplifiedabove, any of a variety of amines can be used in the methods providedherein and known in the art. For example, ethylenediamine,monoethanolamine, tris(hydroxymethyl)aminomethane, alkyl amine, allylamine or any amino modified polymers, including polyethylene glycol(PEG), perfluoropolymers, polystyrene, polyethylene,polydimethylsilixane, polyacrylate, polymethylmethacrylate and the like,and mixtures thereof, can be used. In addition, the linking of thehydrophobic groups, including aliphatic (e.g., hydrocarbons from C₁ toabout C₂₂) groups, aromatic groups, polyethylene polymers, polystyrenepolymers, perfluoropolymers, polydimethylsiloxanes, polyacrylates,polymethylmethacrylates, as well as, hydrophilic groups, including a OHgroup, hydrophilic polymers, such as, PEOX, PEG, PEO etc. to a modifiedABP can be achieved by using, for example, epoxy reactions, amidationreactions, Michael addition reactions, including using a —SH or an —NH₂group reacted with maleimide, aldehyde/ketone-amine/hydrazide couplingreactions, iodo/iodoacetyl-SH coupling reactions,hydroxylamine-aldehyde/ketone coupling reactions etc. Such syntheticstrategies allow not only asymmetric growth of the molecule, where morepockets are introduced, but also the addition of multiple functionalgroups at both the interior and the exterior of the structure. Thehomopolymer can be modified further using the same or a differentsynthetic process until the desired ABP's with appropriate molecularweight and functional groups are attained. In addition, the hydrophobicand hydrophilic properties, as well as charge density of suchhomopolymers, can be tailored to fit specific application needs usingappropriate monomers for constructing the homopolymer and suitablemodification reactions.

In another embodiment of the disclosure, a focal point (merged fromvarious reactive chain ends during a convergent synthesis) of a randomABP, such as, polyoxazoline, can be terminated or reacted with anothersmall molecule to generate various functional groups at thehomopolymeric chain ends, including primary, secondary or tertiaryamines, carboxylate, hydroxyl, alkyl, fluoroalkyl, aryl, PEG, acetate,amide and/or ester groups. Alternatively, various initiators also can beutilized so that the same type of functional group can be introduced atthe surface groups where a polymerization begins during a convergentsynthesis (J. Macromol. Sci. Chem. A9 (5), pp. 703-727 (1975)).

An alkyl surface modified, randomly branched poly(2-ethyloxazoline) witha primary amine group at the focal point of the branched polymer can beprepared using the Litt and Warakomski procedures, supra. For example,CH₃(CH₂)₁₇—Br can be utilized as an initiator for 2-ethyloxazolinepolymerization through a cationic ring opening process to generate arandomly branched polymer, followed by quenching withN-tert-butyloxycarbonylpiperazine (N-Boc-piperazine) or ethylenediamine(EDA). The termination with a large excess of EDA allows thehydrophobically modified branched poly(2-ethyloxazoline) polymer to befunctionalized with a primary amine group at the focal point (FIG. 6B).Alternatively, N-tert-butyloxycarbonylpiperazine (N-Boc-piperazine)terminated hydrophobically modified branched poly(2-ethyloxazoline)polymer also can be deprotected to generate a primary amino group at thefocal point. If not terminated, the focal point of the polymer can behydrolyzed to, for example, a hydroxyl group on dissolving in water(e.g., containing 1N Na₂CO₃).

While the introduction of a primary amine group to a hydrophobicallymodified branched poly(2-oxazoline) homopolymer enhances drug solubilityand produces PAA induced aggregates, the primary amine group also allowsthe attachment of various targeting groups, such as, an antibody,antigen binding portion thereof, an antigen, or a member of a bindingpair to the hydrophobically modified branched poly(2-oxazoline) polymer(FIG. 10). Such aggregates or nanoparticles containing such targetinggroups and modifications thereto can provide a targeting ability on theaggregate with PAA and enables PAA to be released preferentially orsolely at the desired treatment location.

As taught herein, the MBP's, such as, a hydrophobically modifiedhomopolymers, including both SBP's and ABP's, can be used to generate asurface-modified branched polymer for solubilizing water insoluble orpoorly water soluble PAA's, or for forming PAA induced nanoparticleswith water insoluble or poorly water soluble PAA's, such as, paclitaxel,camptothecin, doxorubin, dolargin, loperamide, tubocurarine, ibuprofen,diazepam, naproxen, carbamazepine, griseofulvin, nifedipine,phytosterol, omeprazol, domperidone, zidovudine, amphotericin B and thelike, as well as drugs described herein, and known to be or are modifiedto be poorly soluble in water or insoluble in water. In such a reaction,the hydrophilic or amphiphilic core can be poly(2-oxazoline),poly(2-substituted oxazolines), including poly(2-methyloxazoline,poly(2-ethyloxazoline), poly(2-propyloxazoline) andpoly(2-butyloxazoline) etc., PEG, PEO, polyphosphonate and the like. Thehydrophobic shell can comprise aliphatic hydrocarbons (such as, from C₁to about C₂₂), aromatic hydrocarbons, polyethylene polymers, polystyrenepolymers, perfluoropolymers, polydimethylsiloxanes, polyacrylates,polymethylmethacrylates and the like. On the other hand, asymmetricallybranched PLL, PEI or PEOX homopolymers also can be modified withhydrophobic surface groups listed above to enhance the solubility ofwater insoluble or poorly water soluble PAA's.

The branching density (e.g., from low generation, such as, star and combhomopolymers, to high generation of dendrimers and dendrigrafts), aswell as the amount of hydrophobic surface group coverage (e.g., from 0%to 100% coverage) of the branched homopolymers can affect significantlyhomopolymer solubility, which in turn, also affects the ability todissolve or to adsorb hydrophobic PAA's. For example, the increase inbranching density and the amount of hydrophobic group coverage will makethe homopolymer more compatible with hydrophobic PAA's.

In some cases, the ABP's and SBP's with from about 1 to about 30% ormore surface hydrophobic component by weight are effective atsolubilizing or dispersing poorly water soluble or water insolublePAA's, such as, paclitaxel. In addition, the branched homopolymersutilized, for example, a POX, a PEOX, a PMOX, PEO/PEG, polyacrylamides,polyphosphates, polyvinylpyrrolidones and polyvinyl alcohols are solublein both water and in various organic solvents, thereby facilitatingforming various PAA containing nanoparticles or aggregates. The goodwater solubility along with good hydrophobic drug miscibility in anaqueous solution, with or without other organic solvents, makes suchhomopolymers useful for enhancing the solubility of poorly water solublePAA's. For example, the homopolymers of interest simplify manufacturingprocesses and decrease production cost by reducing formulation steps,processing time, as well as the need to use complex and expensiveequipment currently used in the pharmaceutical industry. If additionalbranching densities are needed, the SBP's or ABP's first can be modifiedwith additional groups as described herein, and then, for example,attached with additional hydrophobic functional groups for enhancing PAAsolubility.

On mixing hydrophobically modified SBP's or ABP's with a water insolubleor poorly water soluble PAA, a distinct physical aggregate is formed ofsize distinct from aggregates formed only of polymer (FIGS. 13-15). Whenthe homopolymer and PAA concentrations decrease, the size anddistribution of the polymer PAA aggregates become much more similar tothat of polymer only aggregates suggesting PAA is released from theinduced aggregates or nanoparticles. The broad size distribution ofpolymer only aggregates is similar to that observed for other structurescomposed of lipid, whether or not associated with a PAA. On the otherhand, the PAA induced aggregates of interest are of a particular size ofnarrower distribution, that is, unique aggregates of certain size areproduced. As PAA concentration in the aggregate decreases, homopolymerconcentration in the aggregate decreases, aggregate concentrationdecreases or any combination thereof, the aggregates of interest releasePAA, as evidenced by a reduction of aggregate size and/or a broaderdistribution of aggregate size. The broader distribution may result froma mixture of homopolymer only aggregates and polymer PAA aggregates ofvarying size due to PAA release, until the only aggregates observed arethose which have the characteristics of those which are homopolymeronly. In other words, the PAA is released gradually after introducedinto a host, such as, in the circulatory system. That mechanism isimportant for various drug delivery applications including, intravenous(IV), oral, transdermal, ocular, intramuscular and the like modes ofadministration, and where a delayed release or sustained release profilemay be desirable.

The PAA induced aggregates also can be linked with a targeting moiety orgroup including, but not limited to, an antibody (or antigen-bindingportion thereof), antigen, cognate carbohydrates (e.g., sialic acid), acell surface receptor ligand, a moiety that binds a cell surfacereceptor, a moiety that binds a cell surface saccharide, anextracellular matrix ligand, a cytosolic receptor ligand, a growthfactor, a cytokine, an incretin, a hormone, a lectin, a lectin target,such as, a galactose, a galactose derivative, an N-acetylgalactosamine,a mannose, a mannose derivative and the like, a vitamin, such as, afolate, a biotin and the like, an avidin, a streptavidin, a neutravidin,a DNA, an RNA etc. to form a conjugate so that the targeting group(s)are incorporated with nanocomposite particle of interest (FIG. 10).

In addition, a diagnostic agent, such as, an imaging agent, aradionuclide or any of a variety of contrasting agents also can becarried by said aggregates of interest. Thus, a combination ofchemotherapy, radiotherapy and/or targeted therapy with real timediagnostic/monitoring capabilities can be achieved (FIGS. 11 and 12). Insome embodiments, the diagnostic agent is poorly soluble or waterinsoluble, thereby negating the need, for example, of a PAA of interestto induce aggregation.

Thus, a diagnostic agent can be a metal containing material or aparamagnetic material, e.g., magnetic resonance imaging materials, whichcan be deposited on the surface or entrapped within a nanocomposite ofinterest so that the nanoparticle can be used as both a diagnostic and atherapeutic agent. In yet another aspect of the disclosure, the imagingmaterial containing nanoparticles further can comprise a targetingmoiety/group, which allows such nanoparticle to target specificlocations that need therapeutic treatment, for example, a tumor site.

Thus, a molecule with the ability to bind another molecule, such as abiological polymer, such as a polypeptide, or a polysaccharide, anenzyme, a receptor and the like, which can bind a vitamin, a lectin, ametal and so on, can be used in a composite of interest. Metals andmetal ions that can be carried by a polymer of interest may include, butare not limited to, transition metals, such as Sc, Y, Ti, Zr, Hf, V, Nb,Ta, Cr, Mo, W, Mn, Tc, Re, Fe, Ru, Os, Co, Rh, Ir, Ni, Pd, Pt, Cu, Ag,Au, Zn, Cd Hg, Ga, In or Tl, alkali metals, alkaline earth metals,Lanthanide series elements, such as Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy,Ho, Er, Tm, Yb and Lu, Actinide series elements, such as Th, Pa, U, Np,Pu, Am, Cm, Bk, Cf, Es, Fm, Md, No and Lr, and the like.

Such can be carried by, for example, one or more chelating groups,including, but not limited to, ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTPA), diethylenetriaminepentaaceticacid (DTPA), 1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA),1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A),1-oxa-4,7,10-triazacyclododecane-triacetic acid (DOXA),1,4,7-triazacyclononanetriacetic acid (NOTA),1,4,8,11-tetraazacyclotetradecanetetraacetic acid (TETA),DOTA-N(2-aminoethyl)amide and DOTA-N-(2-aminophenethyl)amide.

For such diagnostic purposes, a conjugate of interest comprises areporter molecule that can be detected by an external device, such as, agamma camera. Thus, a conjugate can be configured to comprise, forexample, a radioisotope that will emit detectable radiation. Theconjugate is placed into a format suitable for consumption or placementin a body, employing reagents suitable therefor as known in the art. Theconjugate composition is administered as known in the art, such asorally, rectally, intravenously and so on.

In another aspect of the disclosure, the nanoparticle can carrydifferent types of PAA's so that a combination or cocktail therapy canbe achieved. Such PAA's may include, but are not limited to, varioussmall molecule drugs, inorganic drugs and biological molecule baseddrugs, such as, a peptide, a protein, an antibody, an enzyme, a vaccineand the like. The second or more PAA's need not be poorly soluble orwater insoluble as the second or more PAA's can be situated within voidsin the aggregate and need not be located at the surface. Hence, any ofthe PAA's noted herein or known in the art can comprise the second ormore PAA's.

Drug Formulation and Nanoparticle Preparation

PAA and modified homopolymer can be suspended individually in suitablebuffers and/or solvents, such as, a buffer, acetone, ethanol and thelike, at suitable concentrations, such as those which are establishedfor in vivo use, generally in milligram or nanogram quantities. Then,the two solutions are mixed at a suitable temperature, such as, roomtemperature or at another temperature known to be acceptable formaintaining integrity of the PAA and homopolymer, for a suitable periodof time, such as, one hour, two hours and so on. Other incubation timescan vary from minutes to hours as the aggregates of interest are stableonce formed. The aggregates can be concentrated or collected practicingmethods known in the art, for example, by filtration, centrifugation,evaporation, lyophilization, dialysis and the like. The aggregates canbe desiccated for extended shelf life.

For example, paclitaxel was dissolved in ethanol in various amounts ofup to 40 mg/mL. A hydrocarbon (CH₃(CH₂)₁₇) modified randomly branchedPEOX was prepared as taught herein and dissolved at varyingconcentrations of up 100 mg/mL in saline.

The two solutions then were mixed in various volumes to result in finalhomopolymer to paclitaxel molar ratios in the mixtures ranging from 3:1to 10:1. The mixtures subsequently were frozen at −80° C. for 3 hoursthen lyophilized for 20 to 48 hours depending on volume to yield a drypowder.

The size of the aggregates or nanoparticles, as measured by lightscattering, can range from about 120 nm (e.g., at 3 mg paclitaxel permL) to about 165 nm (e.g., at 5 mg paclitaxel per mL) in diameterdepending on the concentration of drug and concentration of homopolymer.

Alternatively, PAA and homopolymer can be dissolved in a common solvent,which generally is not necessarily hydrophilic but is miscible withwater, and then added to an aqueous solution. Hence, paclitaxel and PEOXcan be dissolved in acetone and then dropwise added to water underagitation, such as, while stirred or sonicated, followed by dialysiswith a 1000 MW cutoff membrane. The final product then can belyophilized.

A conjugate of interest can be incorporated into pharmaceuticalcompositions suitable for administration, for example, for diagnosticimaging, for lifestyle management or to attain a therapeutic milestone.Such compositions typically comprise an aggregate of interest and apharmaceutically acceptable carrier, excipient or diluent, which isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like, that is, those ingredients of a pharmaceuticallyacceptable composition aside from the PAA's that are included thereinfor particular purposes, such as, bulking, preservation, delayedrelease, binding and so on, as known in the art, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agents are incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds also can be incorporated into thecomposition.

A pharmaceutical composition of the disclosure for use as disclosedherein is formulated to be compatible with the intended route ofadministration. Examples of routes of administration include parenteral,e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal and rectal administration. Solutionsor suspensions used for parenteral, intradermal or subcutaneousapplication can include a sterile diluent, such as, water for injection,saline, oils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents, such as, benzyl alcohol ormethyl parabens; antioxidants, such as, ascorbic acid or sodiumbisulfite; chelating agents, such as, EDTA; buffers, such as, acetates,citrates or phosphates; and agents for the adjustment of tonicity, suchas, sodium chloride or dextrose. pH can be adjusted with acids or bases,such as HCl or NaOH. The parenteral preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic as an article of manufacture. Generally, an in vivo diagnosticagent will be administered orally, rectally, intravenously,intraperitoneally and so on.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water or phosphate-buffered saline(PBS). The composition generally is sterile and is fluid to the extentthat easy syringability exists. The composition must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as, bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol and the like) and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prevention of theaction of microorganisms can be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal and the like. Isotonic agents, for example,sugars, polyalcohols, such as, mannitol, sorbitol or sodium chloride canbe included in the composition. Prolonged absorption of the injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, aluminum monostearate orgelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount of an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound in a sterile vehicle that contains abasic dispersion medium and the required other ingredients, for example,from those enumerated above, and as known in the art. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreparation can be prepared by, for example, lyophilization, vacuumdrying or freeze drying, that yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof. The preparation of interest can bestored and reconstituted with a suitable liquid for use.

Oral compositions generally include an inert diluent, flavorant, odorantor an edible carrier. The composition can be enclosed in gelatincapsules or compressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches or capsules. Oral compositionsalso can be prepared using a fluid carrier to yield a syrup or liquidformulation, or for use as a mouthwash, wherein the compound in thefluid carrier is applied orally and swished and expectorated orswallowed.

Pharmaceutically compatible binding agents and/or adjuvant materials canbe included as part of the composition. Tablets, pills, capsules,troches and the like can contain a binder, such as, microcrystallinecellulose, gum tragacanth or gelatin; an excipient, such as, starch orlactose, a disintegrating agent, such as, alginic acid, Primogel or cornstarch; a lubricant, such as, magnesium stearate or Sterotes; a glidant,such as, colloidal silicon dioxide; a sweetening agent, such as, sucroseor saccharin; or a flavoring agent, such as, peppermint, methylsalicylate or orange flavoring.

For administration by inhalation, the compound is delivered in the formof, for example, a wet or dry aerosol spray from a pressurized containeror dispenser that contains a suitable propellant, e.g., a gas, such as,carbon dioxide or a nebulizer, or a mist.

Systemic administration also can be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants generally are known in the art and include, for example,for transmucosal administration, detergents, bile salts and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active compounds are formulated into ointments, salves, gels orcreams as generally known in the art. A suitable carrier includesdimethylsulfoxide.

The compound also can be prepared in the form of suppositories (e.g.,with conventional suppository bases, such as, cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compound is prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters and polylactic acid.

Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials also can be obtained commercially, forexample, from Alza Corporation and Nova Pharmaceuticals, Inc.

The instant aggregates can be used in topical forms, such as, creams,ointments, lotions, unguents, other cosmetics and the like. PAA's andother bioactive or inert compounds can be carried, and includeemollients, bleaching agents, antiperspirants, pharmaceuticals,moisturizers, scents, colorants, pigments, dyes, antioxidants, oils,fatty acids, lipids, inorganic salts, organic molecules, opacifiers,vitamins, pharmaceuticals, keratolytic agents, UV blocking agents,tanning accelerators, depigmenting agents, deodorants, perfumes, insectrepellants and the like.

It can be advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for a subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce a desired therapeutic endpoint.

The dosages, for example, preferred route of administration and amountsare obtainable based on empirical data obtained from preclinical andclinical studies, practicing methods known in the art. The dosage anddelivery form can be dictated by and can be dependent on thecharacteristics of the PAA, the polymer, the particular therapeuticeffect to be achieved, the characteristics and condition of therecipient and so on. For repeated administrations over several days orlonger, depending on the condition, the treatment can be sustained untila desired endpoint is attained. An exemplary dosing regimen is disclosedin WO 94/04188.

The progress of the therapy can be monitored by conventional techniquesand assays, as well as patient input.

The pharmaceutical compositions can be included in a container, pack ordispenser together with instructions for administration.

Another method of administration comprises the addition of a compound ofinterest into or with a food or drink, as a food supplement or additive,or as a dosage form taken on a prophylactic basis, similar to a vitamin.The aggregate of interest can be encapsulated into forms that willsurvive passage through the gastric environment. Such forms are commonlyknown, for example, enteric coated formulations. Alternatively, theaggregate of interest can be modified to enhance half life, such as,chemical modification or combination with agents known to result indelayed, sustained or controlled release, as known in the art.

The instant disclosure now will be exemplified in the followingnon-limiting examples.

EXAMPLES Materials

Symmetrically branched PPI dendrimers were purchased from Sigma-Aldrich.Symmetrically branched PEI dendrimers and dendrigrafts were preparedaccording to procedures provided in U.S. Pat. Nos. 4,631,337, 5,773,527,5,631,329 and 5,919,442. All of the antibodies were purchased fromSigma-Aldrich, Biodesign or Fitzgerald. Different generation PAMAMdendrimers were purchased from Dendritech, Inc.

Synthesis of Modified Symmetrically Branched PPIs with Amino FunctionalGroups (m-SB-PPI-NH₂-1.0)

The following reagents including symmetrically branched PPI (SB-PPI-4,8, 16, 32, 64, MW 316, 773, 1,687, 3,514 and 7,168), methyl acrylate(MA, FW=86.09), ethylenediamine (EDA, FW=60.10) and methanol wereutilized.

To a round bottom flask were added 1.0 g PPI-64 dendrimer (MW 7168) and20 ml methanol (solution A). To a separate round bottom flask were added2.4 g methylacrylate (MA) and 10 ml methanol (solution B). Solution Awas then slowly dropped into solution B while stirring at roomtemperature. The resulting solution was allowed to react at 40° C. for 2hours. On completion of the reaction, the solvent and unreacted MAmonomer were removed by rotary evaporation and the product, 2.5 g of MAfunctionalized PPI, then was redissolved in 20 ml of methanol.

To a round bottom flask were added 160 g EDA and 50 ml of methanol,followed by a slow addition of MA functionalized PPI at 0° C. Thesolution then was allowed to react at 4° C. for 48 hours. The solventand the excess EDA were removed by rotary evaporation. The crude productthen was precipitated from an ethyl ether solution and further purifiedby dialysis to give about 2.8 g of primary amine-functionalizedsymmetrically branched PPI (m-SB-PPI-NH₂-1.0) with a molecular weight ofabout 21,760. The product was characterized by ¹H and ¹³C nuclearmagnetic resonance (NMR) and size exclusion chromatography (SEC).

Other MA or primary amine-modified symmetrically branched PPI dendrimersand symmetrically branched PEI dendrigrafts with various molecularweights were prepared in a similar manner.

Synthesis of Modified Symmetrically Branched PPIs with Mixed Hydroxyland Amino Functional Groups (Mix-m-SB-PPI-64-NH₂/OH-2)

Amino functionalized symmetrically branched PPI (m-SB-PPI-64-NH₂-1.0),MA, EDA, monoethanolamine (MEA, FW=61.08) and methanol were utilized.

To a round bottom flask were added 1.0 g amino-modified PPI orm-SB-PPI-NH₂-1.0 produced from the previous procedure and 20 ml ofmethanol (solution A). To a separate round bottom flask were added 2.4 gof MA and 10 ml methanol (solution B). Solution A was then slowlydripped into solution B while stirring at room temperature. Theresulting solution was allowed to react at 40° C. for 2 hours. Oncompletion of the reaction, the solvent and unreacted monomer MA wereremoved by rotary evaporation and the product, 2.5 g of MAfunctionalized m-SB-PPI-64-MA-1.5, then was redissolved in 20 ml ofmethanol.

To a round bottom flask were added 32 g EDA, 130 g MEA and 100 mlmethanol (the mole ratio of EDA:MEA was 20:80), followed by slowaddition of m-SB-PPI-64-MA-1.5 at 0° C. The solution then was allowed toreact at 4° C. for 48 hours. The solvent and the excess EDA were removedby rotary evaporation. The crude product then was precipitated from anethyl ether solution and further purified by dialysis to give about 2.8g of mixed hydroxyl and amino functionalized (mixed surface) SBP(mix-m-SB-PPI-64-NH₂/OH-2.0, with an average of 20% NH₂ and 80% OHsurface groups and a molecular weight of about 21,862).

Other modified random AB-PEI and regular AB PLL molecules with varyingratios of hydroxyl and amino groups, as well as different molecularweights, were prepared in a similar manner.

Random asymmetrically branched PEI's were purchased from Aldrich andPolysciences. Regular ABP's were prepared according to proceduresprovided in U.S. Pat. No. 4,289,872. All of the antibodies werepurchased from Sigma-Aldrich, Biodesign or Fitzgerald.

Synthesis of Modified Random Asymmetrically Branched PEIs with AminoFunctional Groups (m-ran-AB-PEI-NH₂-1.0)

Random asymmetrically branched PEI (ran-AB-PEI, MW 2,000, 25,000 and75,000), MA, EDA and methanol were utilized.

To a round bottom flask were added 1.0 g PEI (MW 2,000) and 20 mlmethanol (solution A). To a separate round bottom flask were added 3.0 gMA and 10 ml methanol (solution B). Solution A was then slowly drippedinto solution B while stirring at room temperature. The resultingsolution was allowed to react at 40° C. for 2 hours. On completion ofthe reaction, the solvent and unreacted MA were removed by rotaryevaporation and the product, MA functionalized PEI, then was redissolvedin 20 ml of methanol.

To a round bottom flask were added 80 g EDA and 50 ml of methanol,followed by a slow addition of MA-functionalized PEI at 0° C. (1 g MAdissolved in 20 ml methanol). The solution then was allowed to react at4° C. for 48 hours. The solvent and excess EDA were removed by rotaryevaporation. The crude product then was precipitated from an ethyl ethersolution and further purified by dialysis to give about 3.0 g of primaryamine-functionalized random asymmetrically branched PEI(m-ran-AB-PEI-NH₂-1.0) with a molecular weight of about 7,300. Theproduct was characterized by ¹H and ¹³C NMR and SEC.

Other MA or primary amine modified random asymmetrically branched PEIand regular asymmetrically branched PLL polymers with various molecularweights were prepared in a similar manner.

Modification of Branched Polymers with Hydrocarbon Chains

The modification of a randomly branched PEI with 10% hydrocarbon chainsis used as an example. One gram of branched PEI (FW=25000) was dissolvedin 10 mL methanol. To the solution were added 0.23 g of 1,2-epoxyhexane(FW=100.16) and the mixture was heated at 40° C. for 2 hours. Thesolvent then was rotary evaporated and the residue redissolved in water.After dialysis (3,500 cutoff), the modified PEI was generated. OtherMBP's, such as, PAMAM, PEI and PPI dendrimers and dendrigrafts, andasymmetric PLL with various percentages and lengths (e.g., C₄, C₁₂, C₁₈and C₂₂) of hydrocarbon chains were prepared in a similar manner.

Synthesis of Modified Random Asymmetrically Branched PEIs with MixedHydroxyl and Amino Functional Groups (m-ran-AB-PEI-NH₂/OH-2)

Amino functionalized random asymmetrically branched PEI(m-ran-AB-PEI-NH₂-1.0), MA, EDA, monoethanolamine (MEA, FW=61.08) andmethanol were utilized.

To a round bottom flask were added 1.0 g amino-modified PEI orm-ran-AB-PEI-NH₂-1.0 produced from the previous procedure and 20 ml ofmethanol (solution A). To a separate round bottom flask were added 3.0 gof MA and 10 ml methanol (solution B). Solution A then was slowlydripped into solution B while stirring at room temperature. Theresulting solution was allowed to react at 40° C. for 2 hours. Oncompletion of the reaction, the solvent and unreacted MA were removed byrotary evaporation and the product, MA functionalizedm-ran-AB-PEI-MA-1.5, then was redissolved in 20 ml of methanol.

To a round bottom flask were added 60 g EDA, 244 g MEA and 100 mlmethanol (the mole ratio of EDA:MEA was 20:80), followed by slowaddition of m-ran-AB-PEI-MA-1.5 at 0° C. (1 g MA dissolved in 20 ml ofmethanol). The solution then was allowed to react at 4° C. for 48 hours.The solvent and excess EDA were removed by rotary evaporation. The crudeproduct then was precipitated from an ethyl ether solution and furtherpurified by dialysis to give about 2.4 g of mixed hydroxyl and aminofunctionalized random ABP (m-ran-AB-PEI-NH₂/OH-2.0, with an average of20% NH₂ and 80% OH surface groups and the molecular weight was about18,000).

Other modified random AB-PEI and regular AB polylysine polymers withvarious ratios of hydroxyl and amino groups, as well as differentmolecular weights were prepared in a similar manner.

Synthesis of Alkyl-Modified Random Asymmetrically BranchedPoly(2-Ethyloxazoline) (PEOX) with Primary Amine Chain End Group

The synthesis of CH₃—(CH₂)₁₁-PEOX-ABP100 (ABP100 is an arbitrary name todenote the ratio of monomer to initiator in the initial reaction) isprovided as a general procedure for the preparation of core shellstructures. A mixture of CH₃(CH₂)₁₁—Br (2.52 g) in 500 ml of toluene wasazeotroped to remove water with a distillation head under N₂ for about15 min. 2-Ethyloxazoline (100 g) was added dropwise through an additionfunnel and the mixture was allowed to reflux between 24 and 48 hours. Oncompletion of the polymerization, 12.12 g of EDA were added to thereactive polymer solution (A) to introduce the amine function group. Themolar ratio of polyoxazoline chain end to EDA was 1 to 20.

N-tert-butyloxycarbonylpiperazine (N-Boc-piperazine) or water (e.g.,with 1N Na₂CO₃) can be added to terminate the reaction. Morpholine orPEI also can be added to the reactive polymer solution (A) to terminatethe reaction. The crude product was redissolved in methanol and thenprecipitated from a large excess of diethyl ether. The bottom layer wasredissolved in methanol and dried by rotary evaporation and vacuum togive an asymmetrically random branched PEOX polymer or PEOX-PEIcopolymer as a white solid (101 g). Other asymmetrically randomlybranched polymers, such as, C₆-PEOX ABP20, 50, 100, 200, 300, 500,C₁₈-PEOX ABP20, 50, 200, 300, 500, C₂₂-PEOX ABP20, 50, 100, 200, 300,500, and polystyrene-PEOX etc., as well as, non-modified and modifiedpoly(2-substituted oxazoline), such as, poly(2-methyloxazoline), wereprepared in a similar manner. All the products were analyzed by SEC andNMR.

Preparation of Mixed Surface Modified Symmetrical Branched Polymer-IgGConjugates

The preparation of mixed surface (OH/NH₂ mix) modified symmetricallybranched PPI-IgG conjugates (mix-m-SB-PPI-64-NH₂/OH-2-IgG conjugates) isprovided as a general procedure for the preparation of polymer antibodyand polymer streptavidin conjugates. Other conjugates, such as,m-SB-PPI-4-NH₂-1-IgG, m-SB-PPI-8-NH₂-1-IgG, m-SB-PPI-16-NH₂-1-IgG,m-SB-PPI-32-NH₂-1-IgG, m-SB-PPI-4-NH₂-2-IgG, m-SB-PPI-8-NH₂-2-IgG,m-SB-PPI-16-NH₂-2-IgG, m-SB-PPI-32-NH₂-2-IgG, m-SB-PPI-4-NH₂-3-IgG,m-SB-PPI-8-NH₂-3-IgG, m-SB-PPI-16-NH₂-3-IgG, m-SB-PPI-32-NH₂-3-IgG,mix-m-SB-PPI-4-NH₂/OH-1 (OH/NH₂ mix)-IgG, mix-m-SB-PPI-8-NH₂/OH-1(OH/NH₂ mix)-IgG, mix-m-SB-PPI-16-NH₂/OH-1 (OH/NH₂ mix)-IgG,mix-m-SB-PPI-32-NH₂/OH-1 (OH/NH₂ mix)-IgG, mix-m-SB-PPI-4-NH₂/OH-2(OH/NH₂ mix)-IgG, mix-m-SB-PPI-8-NH₂/OH-2 (OH/NH₂ mix)-IgG,mix-m-SB-PPI-16-NH₂/OH-2 (OH/NH₂ mix)-IgG, mix-m-SB-PPI-32-NH₂/OH-2(OH/NH₂ mix)-IgG, mix-m-SB-PPI-4-NH₂/OH-3 (OH/NH₂ mix)-IgG,mix-m-SB-PPI-8-NH₂/OH-3 (OH/NH₂ mix)-IgG, mix-m-SB-PPI-16-NH₂/OH-3(OH/NH₂ mix)-IgG, mix-m-SB-PPI-32-NH₂/OH-3 (OH/NH₂ mix)-IgG, as well asprimary amine and mix OH/NH₂ modified combburst PEI dendrigrafts(Generation 0-5) also were obtained in a similar manner. The synthesisof other protein attached to a modified SBP of interest also wasobtained in a similar manner. The biotinylated-IgG conjugates weresynthesized as provided in Bioconjugate Techniques (G. Hermanson,Academic Press, 1996).

LC-SPDP-Mixed Surface m-SB-PPI-64-NH₂/OH-2

To the mixed surface randomly branched mix-m-SB-PPI-64-NH₂/OH-2 (4×10⁻⁷mol) in 400 μl of phosphate buffer (20 mM phosphate and 0.1 M NaCl, pH7.5) were added 4.0×10⁻⁶ mol of sulfo-LC-SPDP (Pierce, Ill.) in 400 μLof water. The mixture was vortexed and incubated at 30° C. for 30minutes. The LC-SPDP-mix-m-SB-PPI-64-NH₂/OH-2 was purified by gelfiltration chromatography and equilibrated with buffer A (0.1 Mphosphate, 0.1 M NaCl and 5 mM EDTA, pH 6.8). The product wasconcentrated further to yield 465 μL of solution with a concentration ofapproximately 0.77 nmol.

Thiolated Mix m-SB-PPI-64-NH₂/OH-2 from LC-SPDP Mix-m-SB-PPI-64-NH₂/OH-2

The LC-SPDP mix-m-SB-PPI-64-NH₂/OH-2 (50 nmol in 65 μl of buffer A) wasmixed with 100 μL of dithiothreitol (DTT) (50 mM in buffer A) and wasincubated at room temperature for 15 minutes. Excess DTT and byproductswere removed by gel filtration with buffer A. The product wasconcentrated in a 10 K Centricon Concentrator to yield 390 μL of thethiolated mix-m-SB-PPI-64-NH₂/OH-2 that was used for conjugation withactivated antibody.

Maleimide R (MAL-R)-Activated Antibody

To the antibody in PBS (310 μL, 5.1 mg or 34 nmol) were added 20.4 μL ofa MAL-R-NHS (N-hydroxysuccinimide) solution (10 mM in water). Themixture was vortexed and incubated at 30° C. for 15 minutes. The productwas purified by gel filtration with buffer A. The maleimide-R-activatedantibody was used for conjugation with the thiolatedmix-m-SB-PPI-64-NH₂/OH-2.

Mix-m-SB-PPI-64-NH₂/OH-2-Antibody Conjugate

To the thiolated mix-m-SB-PPI-64-NH₂/OH-2 (310 μL or 35.7 nmol) wasadded the MAL-R-activated antibody (4.8 mL or 34 nmol). The reactionmixture was concentrated to approximately 800 μL and then allowed toincubate overnight at 4° C. and/or at room temperature for about 1 hr.On completion, the reaction was quenched with 100 μL of ethyl maleimide(50 mmolar solution) and the conjugate then was fractionated on acarboxymethyl cellulose column (5 mL) with a sodium chloride stepgradient in 20 mM phosphate buffer at pH 6. The conjugate was elutedwith a sodium chloride gradient and characterized by cationic exchangechromatography, UV spectroscopy and polyacrylamide gel electrophoresis.

Conjugation Via Reductive Coupling Reduction of Antibody

To the antibody, 2.1 mg or 14 nmol in 160 μL of buffer B (containing 0.1M sodium phosphate, 5 mM EDTA and 0.1 M NaCl, pH 6.0) were added 40 μLof DTT (50 mM in buffer B). The solution was allowed to stand at roomtemperature for 30 min. The product was purified by gel filtration in aSephadex G-25 column equilibrated with buffer B. The reduced antibodywas concentrated to 220 μL and was used for conjugation.

MAL-R-Mixed Surface Modified SBP

To the mixed surface modified SBP in 400 μL (400×10⁻⁹ mols) at pH 7.4were added 400 μL of MAL-R-NHS (10 mM in water). That was mixed andincubated at 30° C. for 15 min. On termination, the product was purifiedon a Sephadex G-25 column equilibrated with buffer B. The MAL-R-mixedsurface modified SBP was collected and stored in aliquots in the samebuffer at −40° C.

Mixed Surface Modified SBP-Antibody Conjugate

To the reduced antibody (14 nmols in 220 μL) was added theMAL-R-mix-m-SB-PPI-64-NH₂/OH-2 (154 μL, 16.6 nmols) with stirring. ThepH was adjusted to about 6.8 by the addition of 12.5 μL of sodiumcarbonate (1.0 M solution), the reaction was continued for 1 hr at roomtemperature and terminated with the addition of 100 μL of cysteamine(0.4 mM solution). The conjugation mixture was purified on a CMcellulose column with a sodium chloride gradient elution.

Preparation of IgG-Asymmetrical Randomly Branched Polymer Conjugates

The preparation of randomly branched mixed surface (OH/NH₂ mix)m-ran-AB-PEI-NH₂/OH-2-IgG conjugates is provided as a general procedurefor the preparation of polymer-antibody and polymer-streptavidinconjugates. Other conjugates such as PEI-IgG, m-ran-AB-PEI-NH₂-1-IgG,m-ran-AB-PEI-NH₂-2-IgG, m-ran-AB-PEI-NH₂-3-IgG, m-ran-AB-PEI-NH₂-4-IgG,as well as m-ran-AB-PEI-NH₂/OH-1 (OH/NH₂ mix)-IgG, m-ran-AB-PEI-NH₂/OH-2(OH/NH₂ mix)-IgG, m-ran-AB-PEI-NH₂/OH-3 (OH/NH₂ mix)-IgG, regularpolylysine polymer, alkyl modified random branchedpoly(2-ethyloxazoline) with primary amine chain ends were allsynthesized in a similar manner. The synthesis of various proteinconjugates with asymmetrically random branched PEOX polymers also isconducted in a similar manner. The biotinylated-IgG conjugates weresynthesized as provided in Bioconjugate Techniques (G. Hermanson,Academic Press, 1996).

LC-SPDP-Mixed Surface m-Ran-AB-PEI-NH₂/OH-2

To the mixed surface randomly branched m-ran-AB-PEI-NH₂/OH-2 (4×10⁻⁷mol) in 400 μL of phosphate buffer (20 mM phosphate and 0.1 M NaCl, pH7.5) were added 4.0×10⁻ mol of sulfo-LC-SPDP (Pierce, Ill.) in 400 μl ofwater. That was vortexed and incubated at 30° C. for 30 minutes. TheLC-SPDP-m-ran-AB-PEI-NH₂/OH-2 was purified by gel filtrationchromatography and equilibrated with buffer A (0.1 M phosphate, 0.1 MNaCl and 5 mM EDTA, pH 6.8). The product was concentrated further toyield 465 μl of solution with a concentration of approximately 0.77nmol/mol.

Thiolated m-Ran-AB-PEI-NH₂/OH-2 from LC-SPDP m-Ran-AB-PEI-NH₂/OH-2

The LC-SPDP m-ran-AB-PEI-NH₂/OH-2 (50 nmol in 65 ml of buffer A) wasmixed with 100 μL of dithiothreitol (DTT) (50 mM in buffer A) and wasallowed to incubate at room temperature for 15 minutes. Excess DTT andbyproducts were removed by gel filtration with buffer A. The product wasconcentrated in a 10 K Centricon Concentrator to yield 390 μL of thethiolated m-ran-AB-PEI-NH₂/OH-2 that was used for conjugation withactivated antibody.

Maleimide-R-activated antibody made as described above was used forconjugation with the thiolated m-ran-AB-PEI-NH₂/OH-2.

m-Ran-AB-PEI-NH₂/OH-2-Antibody Conjugate

To the thiolated m-ran-AB-PEI-NH₂/OH-2 (310 μL or 35.7 nmol) was addedthe MAL-R-activated antibody (4.8 mL or 34 nmol). The reaction mixturewas concentrated to approximately 800 μL and allowed to incubateovernight at 4° C. and/or at room temperature for about 1 hr. Oncompletion, the reaction was quenched with 100 μL of ethyl maleimide (50mmolar solution) and the conjugate then was fractionated on acarboxymethyl cellulose column (5 ml) with a sodium chloride stepgradient in 20 mM phosphate buffer at pH 6. The conjugate was elutedwith a sodium chloride gradient and characterized by cationic exchangechromatography, UV spectroscopy and polyacrylamide gel electrophoresis.

Paclitaxel Formulation and Nanoparticle Preparation

Paclitaxel was dissolved in ethanol to a concentration of up to 40mg/mL.

A C₁₈ hydrocarbon modified randomly branched PEOX was prepared as taughtherein.

The polymer was separately dissolved to a concentration of up to 100mg/mL in saline. The two solutions were then mixed at various volumes toresult in final polymer to paclitaxel molar ratios in the mixturesranging from 3:1 to 10:1. The mixtures were subsequently frozen at −80°C. for 3 hours then lyophilized for 20 to 48 hours depending on volume.

The size of the aggregates as measured by light scattering ranged fromabout 120 nm to about 165 nm in diameter.

Alternatively, both paclitaxel and the PEOX polymer can be dissolved ina common solvent, such as, acetone and then dropwise added to waterwhile being stirred or sonicated, followed by dialysis with a 1000 MWcutoff membrane. The final product then can be generated bylyophilization and the size of the aggregates was measured by lightscattering.

Other PAA induced aggregates or nanoparticles using varioushydrophobically surface modified branched polymers, such as, C₄, C₆, C₁₂or C₂₂ hydrocarbon modified randomly branched PEOX, PEI and PPIpolymers; C₄, C₆, C₁₂, C₁₈ and C₂₂ hydrocarbon modified PAMAM, PEI andPPI dendrimers and dendrigrafts; and C₄, C₆, C₁₂, C₁₈ and C₂₂hydrocarbon modified branched PLL/polymers can be prepared in a similarmanner.

Thus, C₁₈-PEOx-100-NH₂ (500 mg) is dissolved in 5 mL of ethanol to yielda 100 mg/mL solution. A 20 mg/mL solution of Paclitaxel is also preparedby dissolving 100 mg in 5 mL of ethanol. The two solutions are mixed for20 minutes resulting in a solution containing 10 mg Paclitaxel and 50 mgpolymer per mL, providing a solution with a 1:5 drug:polymer ratio. Themixture is placed on a rotary evaporator and the ethanol removed todryness. The resultant solid is redissolved with stirring in 33 mL ofsaline solution to a final Paclitaxel concentration of 3 mg/mL. Thesolution preparation is passed through a 0.8 μm filter and then a 0.22μm filter. The filtrate is frozen in a vial at −70° C. for at least 2hours then lyophilized over a 72 hour period. The vial is stoppered andthe ready-to-use white powder is stored at room temperature.

Nanoparticle Measurement

The size of various polymers, polymer only aggregates, as well asdrug-induced polymer aggregates was measured by a dynamic lightscattering method using a Malvern Zetasizer Nano-ZS Zen3600 particlesize analyzer.

Activity Testing

Metabolism in viable cells produces “reducing equivalents,” such as,NADH or NADPH. Such reducing compounds pass electrons to an intermediateelectron transfer reagent that can reduce the tetrazolium product, MTS(Promega), into an aqueous, soluble formazan product, which is colored.At death, cells rapidly lose the ability to reduce tetrazolium products.The production of the colored formazan product, therefore, isproportional to the number of viable cells in culture.

The CellTiter 96® Aqueous products (Promega) are MTS assays fordetermining the number of viable cells in culture. The MTS tetrazoliumis similar to MTT tetrazolium, with the advantage that the formazanproduct of MTS reduction is soluble in cell culture medium and does notrequire use of a solubilization solution. A single reagent addeddirectly to the assay wells at a recommended ratio of μl reagent to 100μl of culture medium was used. Cells were incubated 1-4 hours at 37° C.and then absorbance was measured at 490 nm.

Thus, the cytotoxicity of various paclitaxel containing aggregates ofinterest, along with commercially available Taxol and Abraxane, apaclitaxel nanoparticle encapsulated with human serum albumin, weretested on different cancer cell lines (from ATCC) including, lung cancerA549, breast cancer MDA-MB-231 and OV 90 ovarian cancer cell lines, aswell as on a normal human fibroblast cell line.

The drug-induced nanoparticles were at least the same or more potent atkilling the cancer cells, particularly at low drug concentrationsranging from 0.5 μg/mL to 0.5 ng/mL. No toxicity to the normal humanfibroblast cell line was observed.

The maximum tolerated dose (MTD) of the drug-induced nanoparticles wascompared to that of Taxol. Over the course of seven weeks, various dosesof Taxol and the paclitaxel-containing nanoparticles of interest wereinjected into the tail vein of CD-1 mice. The MTD of the paclitaxelnanoparticles was more than 7-fold higher than that of Taxol, with nomajor side effects to the surviving mice. Also, significant weight losswas observed in mice receiving Taxol as compared to no weight loss forthe cohort that received the paclitaxel nanoparticles of interest.

A controlled study using lung cancer A548 cell line and breast cancerMDA-MB-231 cell line in a xenograft mouse model revealed that thepaclitaxel nanoparticles inhibited tumor growth in vivo significantlybetter than did Taxol and Abraxane. A rapid reduction of tumors in micereceiving the paclitaxel nanoparticles of interest was observed, ascompared to mice receiving Taxol or Abraxane.

Campothecin Formulation and Nanoparticle Preparation

Camptothecin (2.1 mg) and polymer (10.5 mg) are dissolved in a 4:1 v/vchloroform:ethanol mixture. Following thorough mixing, the solvent isremoved to dryness on a rotary evaporator. The resultant solid mixtureis redissolved in 2 mL of saline solution, mixed, then filtered througha 0.8 um syringe filter. The filtrate is frozen at −70° C. for at least2 hours in a lyophilization vial, then lyophilized overnight (˜16hours). The vial is stoppered and the ready-to-use white powder isstored at −70° C. The material is reconstituted with 2 mL of waterimmediately prior to use.

Irinotecan, also known as CPT-11, is a synthetic analog of camptothecin.CPT-11 contains, for example, a bipiperdine carboxylate group and anethyl group on the A and B rings, to yield a compound with greatercytotoxicity than the parent molecule.

Varying concentrations of CPT-11 and the camptothecin branched polymeraggregates described above were prepared.

Two cancer cell lines were maintained in suitable medium underrecognized culture conditions. MCF-7 is a human breast cancer cell lineand H460 is a human epithelial lung cancer cell line. Those cell lineswere exposed to varying concentrations of CPT-11 and concentrations ofthe camptothecin aggregates based on the amount of drug. Cellsurvivability was assessed as described above.

FIG. 17 summarizes results obtained with the MCF-7 breast cancer cellline. It can be seen that cytotoxicity of the cells to CPT-11 increasedwith increasing drug concentration. On the other hand, the camptothecinaggregate was more highly cytotoxic at all dosages tested. Polymer notassociated with drug was not cytotoxic. Thus, although camptothecin perse does not have the same level of cytotoxicity as does CPT-11, whenaggregated with a branched polymer, that aggregate was more cytotoxicthan CPT-11.

A similar result was obtained with the H460 lung cancer cell line. Asdepicted in FIG. 18, a dose response curve of cytotoxicity to CPT-11 wasobserved. However, the camptothecin/branched polymer aggregates weremore cytotoxic than CPT-11 at each concentration tested.

All references cited herein are herein incorporated by reference inentirety.

It will be appreciated that various changes and modifications can bemade to the teachings herein without departing from the spirit and scopeof the disclosure.

1. The lyophilized aggregate obtained by the method of claim
 16. 2. Themethod of claim 19, wherein said aliphatic chain comprises a branch. 3.(canceled)
 4. The method of claim 16, wherein said surface groupcomprises an aromatic group.
 5. The method of claim 16, wherein saidhydrophobic surface group comprises a saccharide.
 6. The method of claim16, wherein said homopolymer comprises an amine group.
 7. (canceled) 8.The method of claim 16, further comprising in step (a) mixing (iii) atargeting moiety.
 9. (canceled)
 10. The method of claim 16, wherein saidhomopolymer comprises a terminal functional group. 11.-12. (canceled)13. A composition comprising the aggregate of claim 1 and an aqueoussolution.
 14. A composition comprising the aggregate of claim 1 and apharmaceutically acceptable carrier, excipient or diluent. 15.(canceled)
 16. A method of making a lyophilized aggregate comprising:(a) mixing: (i) a water insoluble or a poorly water solublepharmaceutically active agent (PAA) dissolved in a solvent; and (ii) awater soluble randomly branched homopolymer modified with a hydrophobicsurface group, dissolved in a water, a buffer, a saline, an aqueoussolution or an organic solvent; to form an aggregate; and (b)lyophilizing said aggregate to form said lyophilized aggregate, whereinsaid PAA is located at or in said surface group; said lyophilizedaggregate is water soluble; and on dilution in an aqueous solution, saidPAA is released from said lyophilized aggregate at a controlled rate.17. The method of claim 16, wherein said solvent is miscible with water.18. The method of claim 16, wherein said solvent comprises ethanol,acetone or a chloroform/ethanol mixture.
 19. The method of claim 16,wherein said hydrophobic surface group comprises an aliphatic chain. 20.The method of claim 16, wherein said branched homopolymer comprises apolyoxazoline, a poly(2-substituted oxazoline), a polyethyleneglycol, apolyethyleneoxide, a polyacrylamide, a polyphosphate, apolyvinylpyrrolidone, a polyvinyl alcohol, a polyethyleneimine, apolypropyleneimine, or a polyamidoamine.
 21. The method of claim 16,wherein said hydrophobic surface group comprises aliphatic and/orsaturated or unsaturated hydrocarbons comprising from 1 to about 22carbons, aromatic hydrocarbons or a combination thereof.
 22. The methodof claim 16, wherein said water insoluble or poorly water soluble PAAcomprises an antiinfective agent or an antineoplastic agent.
 23. Themethod of claim 16, wherein said water insoluble or poorly water solublePAA comprises paclitaxel, docetaxel, taxotere, vinblastine, vincristine,vindesine, vinorelbine, irinotecan, topotecan, camptothecin,camptothecin derivatives (such as, irinotecan, topotecan etc.),doxorubin, cisplatin, carboplatin, oxaliplatin, satraplatin, dolargin,loperamide, tubocurarine, ibuprofen, diazepam, naproxen, carbamazepine,griseofulvin, nifedipine, phytosterol, omeprazol, domperidone,zidovudine, amphotericin B, chlormethine, chlorambucil, busulfan,thiotepa, cyclophosphamide, estramustine, ifosfamide, meclilorethamine,melphalan, uramustine, lonuistine, streptozotocin, dacarbazine,procarbazine, temozolainide,(SP-4-3)-(cis)-aminedichloro-[2-methylpyridine]-platinum (II),methotrexate, permetrexed, raltitrexed, trimetrexate, cladribine,chlorodeoxyadenosine, clofarabine, fludarabine, mercaptopurine,pentostatin, thioguanine, azacitidine, capecitabine, cytarabine,edatrexate, floxuridine, 5-fluorouracil, gemcitabine, troxacitabine,bleomycin, dactinomycin, adriamycin, actinomycin, mithramycin,mitomycin, mitoxantrone, porfiromycin, daunorubicin, epirubicin,idarubicin, valrubicin, phenesterine, tamoxifen, piposulfancamptothesin,amsacrine, etoposide, teniposide, fluoxymesterone, testolactone,bicalutamide, cyproterone, flutamide, nilutamide, aminoglutethimide,anastrozole, exemestane, formestane, letrozole, dexamethasone,prednisone, diethylstilbestrol, fulvestrant, raloxifene, toremifene,buserelin, goserelin, leuprolide, triptorelin, medroxyprogesteroneacetate, megestrol acetate, levothyroxine, liothyronine, altretamine,levamisole, mitotane, octreotide, procarbazine, suramin, thalidomide,methoxsalen, sodium porfimer, bortezomib, erlotinib hydrochloride,gefitinib, imatinib mesylate, semaxanib, adapalene, bexarotene,trans-retinoic acid, 9-cis-retinoic acid and N-(4-hydroxyphenyl)retinamide or a combination thereof.
 24. The method of claim 20, whereinsaid poly(2-substituted oxazoline) comprises poly(2-methyloxazoline,poly(2-ethyloxazoline), poly(2-propyloxazoline) orpoly(2-butyloxazoline).
 25. The lyophilized aggregate of claim 1 whichis water soluble.
 26. The lyophilized aggregate of claim 25 which ondilution in an aqueous solution, said PAA is released from saidaggregate at a controlled rate.